RNA-guided nucleases and active fragments and variants thereof and methods of use

Abstract

The present invention provides compositions and methods for binding a target sequence of interest. The compositions can be used to cleave or modify a target sequence of interest, to visualize a target sequence of interest, and to modify the expression of a sequence of interest. Compositions comprise an RNA-guided nuclease (RGN) polypeptide, a CRISPR RNA, a trans-activated CRISPR RNA, a guide RNA, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided is an RGN system for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 371,230, filed on August 12, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically as an XML file

[0004] This application contains a sequence list, which has been submitted in xml format through the USPTO Patent Center and is now incorporated herein by reference in its entirety. The xml copy was created on August 11, 2023, named L103438_1310WO_Seq_List.xml, and is 1.49MB in size. Field of the Invention

[0005] The present invention relates to the fields of molecular biology and gene editing. Background of the Invention

[0007] Targeted genome editing or modification is rapidly becoming an important tool for basic and applied research. The initial method involves engineered nucleases, such as meganucleases, zinc finger fusion proteins or TALENs, which need to produce chimeric nucleases with engineered, programmable, sequence-specific DNA binding domains specific for each specific target sequence. RNA-guided nucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)-related (Cas) proteins of the CRISPR-Cas bacterial system allow targeting specific sequences by combining nucleases with guide RNAs that specifically hybridize with specific target sequences. Producing target-specific guide RNAs is less expensive and more efficient than producing chimeric nucleases for each target sequence. This RNA-guided nuclease can be used to edit the genome, optionally by introducing sequence-specific double-strand breaks, which can be repaired by error-prone non-homologous end joining (NHEJ) to introduce mutations at specific genomic locations. Alternatively, heterologous DNA can be introduced into the genomic site by homology-directed repair. When fused with a deaminase, RNA-guided nucleases (RGNs) can also be used for base editing. Summary of the invention

[0008] Compositions and methods for binding to a target sequence of interest in a target nucleic acid molecule are provided. The composition can be used to cut or modify a target nucleic acid molecule of interest, detect a target sequence of interest, and modify the expression of a gene of interest comprising a target sequence. The composition comprises RNA-guided nuclease (RGN) polypeptides, CRISPR RNA (crRNA), trans-activated CRISPR RNA (tracrRNA), guide RNA (gRNA) such as a single guide RNA (sgRNA), nucleic acid molecules encoding it, compositions comprising it, and vectors and host cells comprising the nucleic acid molecules. An RGN system and a ribonucleoprotein complex for binding to a target sequence of interest are also provided, wherein the RGN system and the ribonucleoprotein complex comprise RNA-guided nuclease polypeptides and one or more guide RNAs. Therefore, the method disclosed herein is used to bind to a target sequence of interest in a target nucleic acid molecule, and in some embodiments, to cut or modify a target nucleic acid molecule of interest. For example, due to non-homologous end connection, homology-directed repair or base editing of the introduced donor sequence can modify the target nucleic acid molecule of interest.

[0009] In one aspect, the disclosure provides a nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20.

[0010] In some embodiments of the above aspects, when the RGN polypeptide is bound to a guide RNA (gRNA) that is capable of hybridizing to a non-target strand of a target sequence, the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided, sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand.

[0011] In some embodiments of the above aspects, the polynucleotide encoding the RGN polypeptide is operably linked to a promoter that is heterologous to the polynucleotide.

[0012] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having 95% sequence identity to any one of SEQ ID NOs: 1-20. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-20.

[0013] In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding. In some embodiments, the RGN polypeptide is capable of producing a double-strand break. In some embodiments, the RGN polypeptide is capable of producing a single-strand break.

[0014] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or is a nickase.

[0015] In some embodiments of the above aspects, the RGN polypeptide is operably fused to a base editing polypeptide. In some embodiments, the base editing polypeptide is a deaminase, such as a cytosine deaminase or an adenine deaminase. In some embodiments, the deaminase has at least 90% or 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 481-552.

[0016] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0017] In some embodiments of the above aspects, the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0018] In some embodiments of the above aspects, the target sequence is located adjacent to a protospacer adjacent motif (PAM).

[0019] In another aspect, the present disclosure provides a vector comprising a nucleic acid molecule as described above.

[0020] In some embodiments of the above aspects, the vector further comprises at least one nucleotide sequence encoding the gRNA, which is capable of hybridizing to the non-target strand of the target sequence.

[0021] In some embodiments of the above aspects, the guide RNA is selected from the group consisting of: a) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:21; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:1; b) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:22; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:43; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:2; c) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:23; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:3; d) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:45; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:3 NO:4 has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity; e) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042;wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:5; a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:26; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:6; g) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NO:1044 or 1045; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; NO:48 or having at least 90%, at least 95%, or 100% sequence identity to nucleotides 27-96 of SEQ ID NO:1044 or nucleotides 27-95 of SEQ ID NO:1045; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7; h) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:28; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:49; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:8; i) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:28; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:49; NO:29 having at least 90%, at least 95% or 100% sequence identity; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:50; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:9; j) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:30; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:51;wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 10; k) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 31; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 52; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 11; l) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 32; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 53; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:12 having at least 90%, at least 95% or 100% sequence identity; m) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:33; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:54; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:13; n) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:34; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:55; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: NO:14 having at least 90%, at least 95% or 100% sequence identity; o) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:35; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:15;p) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:36; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:57; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:16; q) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:37; and ii) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:58; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:17; r) a guide RNA comprising: i) a CRISPR and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:59 or 60; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:18; s) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:40; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:61; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:19; and t) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:40; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:61; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:19; NO:41 has at least 90%, at least 95% or 100% sequence identity to a CRISPR repeat sequence; and ii) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:62; wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:20. ;

[0022] In some embodiments of the above aspects, the gRNA is a single guide RNA. In some embodiments of the above aspects, the gRNA is a dual guide RNA.

[0023] On the other hand, the disclosure provides cells comprising the nucleic acid molecules or vectors described above. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or a bird cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0024] In another aspect, the present disclosure provides a plant or seed comprising the plant cell described above.

[0025] In another aspect, the disclosure provides a method of preparing a RGN polypeptide, comprising culturing the above-described cell under conditions where the RGN polypeptide is expressed.

[0026] In another aspect, the disclosure provides a method of making an RGN polypeptide, comprising introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; and culturing the cell under conditions whereby the RGN polypeptide is expressed.

[0027] In some embodiments of the above aspects, when the RGN polypeptide is bound to a guide RNA (gRNA) that is capable of hybridizing to a non-target strand of a target sequence, the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided, sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand.

[0028] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having at least 95% or 100% sequence identity to any one of SEQ ID NOs: 1-20.

[0029] In some embodiments of the above aspects, the method further comprises purifying the RGN polypeptide.

[0030] In some embodiments of the above aspects, the cell further expresses one or more guide RNAs capable of binding to the RGN polypeptide to form an RGN ribonucleoprotein complex. In some embodiments, the method further comprises purifying the RGN ribonucleoprotein complex.

[0031] In another aspect, the present disclosure provides an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20.

[0032] In some embodiments of the above aspects, when the RGN polypeptide is bound to a guide RNA (gRNA) that is capable of hybridizing to a non-target strand of a target sequence, the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided, sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand.

[0033] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having at least 95% or 100% sequence identity to any one of SEQ ID NOs: 1-20.

[0034] In some embodiments of the above aspects, the RGN polypeptide is an isolated RGN polypeptide.

[0035] In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding. In some embodiments, cleavage by the RGN polypeptide produces a double-strand break. In some embodiments, cleavage by the RGN polypeptide produces a single-strand break.

[0036] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or is a nickase.

[0037] In some embodiments of the above aspects, the RGN polypeptide is operably fused to a base editing polypeptide. In some embodiments, the base editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or an adenine deaminase. In some embodiments, the deaminase has at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 481-552.

[0038] In some embodiments of the above aspects, the target sequence is located adjacent to a protospacer adjacent motif (PAM).

[0039] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0040] In another aspect, the present disclosure provides a ribonucleoprotein (RNP) complex comprising the RGN polypeptide described above and a guide RNA bound to the RGN polypeptide.

[0041] In another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) or a polynucleotide encoding a crRNA, wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, wherein the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 21-41, or nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045.

[0042] In some embodiments of the above aspects, the guide RNA comprises the crRNA and a trans-activating CRISPR RNA (tracrRNA) that hybridizes to the CRISPR repeat sequence of the crRNA, and when the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide, the guide RNA is capable of hybridizing to a non-target strand of a target sequence in a target nucleic acid molecule through the spacer sequence of the crRNA in a sequence-specific manner.

[0043] In some embodiments of the above aspects, the polynucleotide encoding the crRNA is operably linked to a promoter heterologous to the polynucleotide.

[0044] In some embodiments of the above aspects, the CRISPR repeat sequence comprises a nucleotide sequence having at least 95% or 100% sequence identity to any one of SEQ ID NOs: 21-41, or nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045.

[0045] On the other hand, the present disclosure provides a vector comprising a nucleic acid molecule comprising a polynucleotide encoding the crRNA described above.

[0046] In some embodiments of the above aspects, the vector further comprises a polynucleotide encoding the tracrRNA. In some embodiments, the tracrRNA is selected from the group consisting of: a) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 42, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21; b) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 43, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 22; c) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 44 or to nucleotides 19-111 of SEQ ID NO: 1040, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 23; d) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 44 or to nucleotides 19-111 of SEQ ID NO: 1040, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 23; NO: 1041 or 1042, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042; f) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 47 or to nucleotides 24-138 of SEQ ID NO: 1043, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 24; e) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042. g) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO: 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045;h) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:49, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:28; i) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:50, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:29; j) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:30; k) a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:52, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:31 has at least 90%, at least 95% or 100% sequence identity; l) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:53, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32; m) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:54, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:33; n) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:55, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:34; o) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:37; NO:56 having at least 90%, at least 95% or 100% sequence identity, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:35; p) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:36; q) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:58, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:37;r) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 59 or 60, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 38 or 39; s) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 61, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 40; and t) a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 62, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 41. ;

[0047] In some embodiments of the above aspects, the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to the same promoter and are encoded as a single guide RNA.

[0048] In some embodiments of the above aspects, the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to separate promoters.

[0049] In some embodiments of the above aspects, the vector further comprises a polynucleotide encoding the RGN polypeptide. In some embodiments, the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 1, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 42; b) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 2, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 22, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 43; c) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 3, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 44. NO:23 has at least 90%, at least 95%, or 100% sequence identity, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:4, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:45; e) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:5, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:25 or to SEQ ID NO: 1041 or 1042 has at least 90%, at least 95%, or 100% sequence identity to nucleotides 1-17, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042;f) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:6, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:26, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NOs:1044 or 1045, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:48 or nucleotides 27-96 of SEQ ID NO:1044 or 1045. NO:1045 has at least 90%, at least 95%, or 100% sequence identity to nucleotides 27-95 of SEQ ID NO:1045; h) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:8, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:28, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:49; i) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:9, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:29, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:50; j) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:8, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:29, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:50; NO:10 an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:30, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51; k) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:11, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:31, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:52;l) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 32, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 53; m) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 13, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 33, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 54; n) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 14, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:34 has at least 90%, at least 95% or 100% sequence identity, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:55; o) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:15, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:35, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56; p) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:16, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:36, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57; q) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:15, wherein the CRISPR repeat sequence has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:36, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57; ID NO:17 has at least 90%, at least 95%, or 100% sequence identity to a RGN polypeptide, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:37, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:58;r) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 18, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 38 or 39, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 59 or 60; s) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 19, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 40, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 61; and t) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 20, wherein the CRISPR repeat sequence has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 38 or 39, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 59 or 60. NO:41 has at least 90%, at least 95% or 100% sequence identity, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity with SEQ ID NO:62. ;

[0050] In another aspect, the present disclosure provides a nucleic acid molecule comprising a trans-activating CRISPR RNA (tracrRNA) or a polynucleotide encoding a tracrRNA, wherein the polynucleotide encoding the tracrRNA comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NO: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045.

[0051] In some embodiments of the above aspects, the guide RNA comprises the tracrRNA and a crRNA comprising a spacer sequence and a CRISPR repeat sequence, wherein the tracrRNA hybridizes to the CRISPR repeat sequence of the crRNA, and when the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide, the guide RNA is capable of hybridizing to a non-target strand of a target sequence in a target nucleic acid molecule through the spacer sequence of the crRNA in a sequence-specific manner.

[0052] In some embodiments of the above aspects, the polynucleotide encoding the tracrRNA is operably linked to a promoter that is heterologous to the polynucleotide.

[0053] In some embodiments of the above aspects, the tracrRNA comprises a nucleotide sequence having at least 95% or 100% sequence identity to any one of SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NOs: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045.

[0054] In another aspect, the present disclosure provides a vector comprising a nucleic acid molecule comprising a polynucleotide encoding the tracrRNA described above.

[0055] In some embodiments of the above aspects, the vector further comprises a polynucleotide encoding the crRNA. In some embodiments, the crRNA comprises a CRISPR repeat sequence selected from the group consisting of: a) a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21, wherein the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 42; b) a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 22, wherein the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 43; c) a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 23, wherein the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 44 or to nucleotides 19-111 of SEQ ID NO: 1040; d) a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 2 1041 or 1042, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 24; wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 45; e) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; f) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 26, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 47 or to SEQ ID NO: 1041 or 1042. g) a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to nucleotides 24-138 of SEQ ID NO: 1043; g) a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045, wherein the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045;h) a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49; i) a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:50; j) a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:30, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:51; k) a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:31, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: NO:52 has at least 90%, at least 95% or 100% sequence identity; l) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:53; m) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:33, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:54; n) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:34, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:55; o) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:34, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:55; NO:35 has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56; p) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:36, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57; q) a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:37, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:58;r) a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:38 or 39, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:59 or 60; s) a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:40, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:61; and t) a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:41, wherein the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:62. ;

[0056] In some embodiments of the above aspects, the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to the same promoter and are encoded as a single guide RNA.

[0057] In some embodiments of the above aspects, the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to separate promoters.

[0058] In some embodiments of the above aspects, the vector further comprises a polynucleotide encoding the RGN polypeptide. In some embodiments, the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 1, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 42; b) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 2, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 22, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 43; c) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 3, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 44. NO:23 has at least 90%, at least 95%, or 100% sequence identity, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:4, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:45; e) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:5, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:25 or to SEQ ID A CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, and the tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042;f) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:6, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:26, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NOs:1044 or 1045, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:48 or nucleotides 27-96 of SEQ ID NO:1044 or SEQ ID NO:1045. NO:1045 has at least 90%, at least 95% or 100% sequence identity to nucleotides 27-95 of SEQ ID NO:1045; h) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49; i) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:9, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:50; j) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: NO:10 A RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:30, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51;k) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 11, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 31, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 52; l) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 12, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 32, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 53; m) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 13, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:33 has at least 90%, at least 95% or 100% sequence identity, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:54; n) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:14, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:34, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:55; o) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:15, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:35, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: NO:56 has at least 90%, at least 95%, or 100% sequence identity; p) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:16, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:36, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:57;q) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 17, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 37, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58; r) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 18, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 38 or 39, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 59 or 60; s) an RGN polypeptide having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 19, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 37, and the tracrRNA has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58; NO:40 has at least 90%, at least 95% or 100% sequence identity, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:61; and t) an RGN polypeptide having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:20, wherein the crRNA comprises a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:41, and the tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:62.;

[0059] In another aspect, the present disclosure provides a cell comprising the nucleic acid molecule, vector, single guide RNA or dual guide RNA described above.

[0060] In some embodiments of the above aspects, the cell is a prokaryotic cell.

[0061] In some embodiments of the above aspects, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or an avian cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0062] In another aspect, the present disclosure provides a plant or seed comprising the plant cell described above.

[0063] In another aspect, the present disclosure provides a system for binding a target sequence in a target nucleic acid molecule, wherein the target sequence comprises a target strand and a non-target strand, the system comprising: a) one or more guide RNAs capable of hybridizing to the non-target strand of the target sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20, or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide; wherein the one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target sequence.

[0064] In some embodiments of the above aspects, at least one of the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the RGN polypeptide is operably linked to a promoter that is heterologous to the nucleotide sequence.

[0065] In another aspect, the present disclosure provides a system for binding a target sequence in a target nucleic acid molecule, wherein the target sequence comprises a target strand and a non-target strand, the system comprising: a) one or more guide RNAs capable of hybridizing to the non-target strand of the target sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; wherein the one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target sequence.

[0066] In some embodiments of the above aspects, at least one of the nucleotide sequences encoding the one or more guide RNAs is operably linked to a promoter that is heterologous to the nucleotide sequence.

[0067] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having at least 95% or 100% sequence identity to any one of SEQ ID NOs: 1-20.

[0068] In some embodiments of the above aspects, the RGN polypeptide and the one or more guide RNAs are not found complexed with each other in nature.

[0069] In some embodiments of the above aspects, the target sequence is a eukaryotic target sequence.

[0070] In some embodiments of the above aspects, the gRNA is a single guide RNA (sgRNA).

[0071] In some embodiments of the above aspects, the gRNA is a dual-guide RNA.

[0072] In some embodiments of the above aspects, the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:1; b) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:43, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:2; c) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:23 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:3; d) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:45, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:4; e) a gRNA comprising an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:25 or to SEQ ID a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5;f) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 26 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 47 or to nucleotides 24-138 of SEQ ID NO: 1043, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6; g) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6; NO:7 having at least 90%, at least 95% or 100% sequence identity; h) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:50, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8; NO:30 having at least 90%, at least 95%, or 100% sequence identity to a CRISPR repeat sequence and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:10;k) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:31 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:52, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 11; l) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:32 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:53, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 12; m) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:33 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:54, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:13; n) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:34 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:55, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:14; o) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:35 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:56, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:13; NO:15 having at least 90%, at least 95%, or 100% sequence identity; p) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:36 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:57, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:16;q) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 17; r) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 59 or 60, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 18; s) a gRNA comprising NO:40 having at least 90%, at least 95%, or 100% sequence identity and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:61, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:19; and t) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:62, wherein the RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:20.;

[0073] In some embodiments of the above aspects, the target sequence is located adjacent to a protospacer adjacent motif (PAM).In some embodiments, the target sequence is intracellular.

[0074] In some embodiments of the above aspects, wherein the one or more guide RNAs are capable of hybridizing to the non-target strand of the target sequence, and the guide RNAs are capable of forming a complex with the RGN polypeptide to guide cleavage of the target nucleic acid molecule.

[0075] In some embodiments of the above aspects, the cleavage produces a double-stranded break.

[0076] In some embodiments of the above aspects, the cleavage produces a single strand break.

[0077] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or is a nickase.

[0078] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a base editing polypeptide. In some embodiments, the base editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or an adenine deaminase. In some embodiments, the deaminase has at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 481-552.

[0079] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0080] In some embodiments of the above aspects, the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0081] In some embodiments of the above aspects, the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the RGN polypeptide are located on one vector.

[0082] In some embodiments of the above aspects, the system further comprises one or more donor polynucleotides.

[0083] In another aspect, the present disclosure provides a cell comprising the system described above.

[0084] In some embodiments of the above aspects, the cell is a prokaryotic cell.

[0085] In some embodiments of the above aspects, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or an avian cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0086] In another aspect, the present invention provides a plant or seed comprising the plant cell described above.

[0087] In another aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid molecule, a vector, a cell, an RGN polypeptide, an RNP complex or the system described above, and a pharmaceutically acceptable carrier.

[0088] In some embodiments of the above aspects, the pharmaceutically acceptable carrier is heterologous to the nucleic acid molecule, the vector, the cell, the RGN polypeptide, or the system.

[0089] In some embodiments of the above aspects, the pharmaceutically acceptable carrier is not naturally occurring.

[0090] In some embodiments of the above aspects, the pharmaceutical composition is lipid-based. In some embodiments, the lipid-based pharmaceutical composition comprises liposomes or lipid nanoparticles (LNP). In some embodiments, the nucleic acid molecule, carrier, cell, RGN polypeptide, RNP complex or system is encapsulated in liposomes or LNP, and / or non-covalently or covalently attached to liposomes or LNP.

[0091] In another aspect, the present disclosure provides a method for binding to a target sequence in a target nucleic acid molecule, comprising delivering the system described above to the target sequence or a cell comprising the target sequence.

[0092] In some embodiments of the above aspects, the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target sequence.

[0093] In some embodiments of the above aspects, the guide RNA or the RGN polypeptide further comprises an expression regulator, thereby regulating the expression of a target gene comprising the target sequence.

[0094] In another aspect, the present disclosure provides a method for cleaving and / or modifying a target nucleic acid molecule comprising a target sequence, the method comprising delivering the system described above to the target sequence or a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

[0095] In some embodiments of the above aspects, the modified target nucleic acid molecule comprises an insertion of heterologous DNA into the target DNA sequence.

[0096] In some embodiments of the above aspects, the modified target nucleic acid molecule comprises a deletion or mutation of at least one nucleotide in the target nucleic acid molecule.

[0097] In another aspect, the disclosure provides a method for binding a target sequence in a target nucleic acid molecule, wherein the target sequence comprises a target strand and a non-target strand, wherein the method comprises: a) assembling an RNA-guided nuclease (RGN) ribonucleotide complex under conditions suitable for forming the RGN ribonucleotide complex by combining: i) one or more guide RNAs capable of hybridizing to the non-target strand of the target sequence; and ii) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; and b) contacting the target nucleic acid molecule or a cell comprising the target nucleotide molecule with the assembled RGN ribonucleotide complex; wherein the one or more guide RNAs hybridize to the non-target strand of the target sequence, thereby directing the RGN polypeptide to bind to the target sequence.

[0098] In some embodiments of the above aspects, the method is performed in vitro, in vivo, or ex vivo.

[0099] In some embodiments of the above aspects, the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target sequence.

[0100] In some embodiments of the above aspects, the guide RNA or the RGN polypeptide further comprises an expression regulator, thereby allowing for said regulating expression of a target gene comprising the target sequence.

[0101] In some embodiments of the above aspects, the RGN polypeptide further comprises a base editing polypeptide, thereby allowing modification of the target nucleic acid molecule. In some embodiments, the base editing polypeptide comprises a deaminase. In some embodiments, the deaminase is a cytosine deaminase or an adenine deaminase. In some embodiments, the deaminase has at least 90%, at least 95%, or 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 481-552.

[0102] In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving a target nucleic acid molecule, thereby allowing for cleavage and / or modification of the target nucleic acid molecule.

[0103] In another aspect, the disclosure provides a method for cleaving and / or modifying a target nucleic acid molecule comprising a target sequence, wherein the target sequence comprises a target strand and a non-target strand, wherein the method comprises contacting the target nucleic acid molecule with: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; and b) one or more guide RNAs capable of targeting the RGN of (a) to the target sequence; wherein the one or more guide RNAs hybridize to the non-target strand of the target sequence, thereby directing the RGN polypeptide to bind to the target nucleic acid molecule, and cleavage and / or modification of the target nucleic acid molecule occurs.

[0104] In some embodiments of the above aspects, double-stranded breaks are produced by cleavage of the RGN polypeptide.

[0105] In some embodiments of the above aspects, single-strand breaks are generated by cleavage of the RGN polypeptide.

[0106] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or a nickase and is operably fused to a base editing polypeptide. In some embodiments, the base editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or an adenine deaminase. In some embodiments, the deaminase has at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 481-552.

[0107] In some embodiments of the above aspects, the modified target nucleic acid molecule comprises an insertion of heterologous DNA into the target nucleic acid molecule.

[0108] In some embodiments of the above aspects, the modified target nucleic acid molecule comprises a deletion or mutation of at least one nucleotide in the target nucleic acid molecule.

[0109] In some embodiments of the above aspects, the target sequence is located adjacent to a protospacer adjacent motif (PAM).

[0110] In some embodiments of the above aspects, the target sequence is a eukaryotic target sequence.

[0111] In some embodiments of the above aspects, the gRNA is a single guide RNA (sgRNA).

[0112] In some embodiments of the above aspects, the gRNA is a dual-guide RNA.

[0113] In some embodiments of the above aspects, the RGN comprises an amino acid sequence having at least 95% or 100% sequence identity to any one of SEQ ID NOs: 1-20.

[0114] In some embodiments of the above aspects, a) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 1, and the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 42; b) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 2, and the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 22 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 43; c) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 3, and the guide RNA comprises a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 44. NO:23 has at least 90%, at least 95% or 100% sequence identity and a tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:4, and the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:45; e) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:5, and the guide RNA comprises a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:25 or to SEQ ID NO: 1041 or 1042 has at least 90%, at least 95%, or 100% sequence identity to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; f) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6, and the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 26, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 47 or to nucleotides 24-138 of SEQ ID NO: 1043;g) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:7, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NO:1044 or 1045, and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO:1045; h) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28, and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49; i) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: ID NO:9 has at least 90%, at least 95% or 100% sequence identity, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:50; j) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:10, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:30 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:51; k) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:11, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: ID NO:31 has a crRNA repeat sequence with at least 90%, at least 95% or 100% sequence identity and a tracrRNA with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:52; 1) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:12, and the guide RNA comprises a crRNA repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32 and a tracrRNA with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:53;m) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 13, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 33, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 54; n) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 14, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 34, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 55; o) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 15, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: ID NO:35 has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:35, and a tracrRNA has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56; p) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:16, and the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:36, and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57; q) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:17, and the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:37, and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57 NO:58 has at least 90%, at least 95% or 100% sequence identity to the tracrRNA; r) the RGN has at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:18, and the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:38 or 39 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:59 or 60;s) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 19, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 40, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 61; and t) the RGN has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 20, the guide RNA comprises a crRNA repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 41, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 62. ;

[0115] In some embodiments of the above aspects, the target sequence is within a cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or an avian cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0116] In some embodiments of the above aspects, the method further comprises culturing the cell under conditions where the RGN polypeptide is expressed, and cleaving and modifying the target nucleic acid molecule to produce a modified target nucleic acid molecule; and selecting a cell comprising the modified target nucleic acid molecule.

[0117] In another aspect, the present disclosure provides a cell comprising the modified target nucleic acid molecule described above.

[0118] In some embodiments of the above aspects, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or an avian cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0119] In another aspect, the present disclosure provides a plant or seed comprising the plant cell described above.

[0120] In another aspect, the present disclosure provides a pharmaceutical composition comprising the cell described above and a pharmaceutically acceptable carrier.

[0121] In another aspect, the present disclosure provides a method for producing a genetically modified cell having a correction in a causal mutation of a genetically inherited disease, the method comprising introducing into the cell: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20, or a polynucleotide encoding the RGN polypeptide, wherein the polynucleotide encoding the RGN polypeptide is operably linked to a promoter to enable expression of the RGN polypeptide in the cell; and b) a guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell; whereby the RGN and gRNA target the genomic location of the causal mutation and modify the genomic sequence to remove the causal mutation.

[0122] In some embodiments of the above aspects, the RGN is nuclease-inactivated or a nickase and is fused to a polypeptide having base editing activity. In some embodiments, the base editing polypeptide is a deaminase. In some embodiments, the polypeptide having base editing activity is a cytosine deaminase or an adenine deaminase. In some embodiments, the deaminase has at least 90%, at least 9%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 481-552.

[0123] In some embodiments of the above aspects, the genetic disease is caused by a single nucleotide polymorphism.

[0124] In some embodiments of the above aspects, the genetic inherited disease is Hurler syndrome.

[0125] In some embodiments of the above aspects, the gRNA further comprises a spacer sequence targeting the proximal region of the causal single nucleotide polymorphism.

[0126] In another aspect, the present disclosure provides a method for generating a genetically modified cell having a deletion in a pathogenic expanded trinucleotide repeat sequence, the method comprising introducing into the cell: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises a sequence corresponding to SEQ ID NO: 1-20 has an amino acid sequence with at least 90% sequence identity, or a polynucleotide encoding the RGN polypeptide, wherein the polynucleotide encoding the RGN polypeptide is operably linked to a promoter to enable the RGN polypeptide to be expressed in the cell; and b) a first guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable the gRNA to be expressed in the cell, and further wherein the gRNA comprises a spacer sequence targeting the 5' flank of the expanded trinucleotide repeat sequence; and c) a second guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable the gRNA to be expressed in the cell, and further wherein the second gRNA comprises a spacer sequence targeting the 3' flank of the expanded trinucleotide repeat sequence; thereby the RGN and the two gRNAs are targeted to the expanded trinucleotide repeat sequence, and at least a portion of the expanded trinucleotide repeat sequence is removed.

[0127] In some embodiments of the above aspects, the genetic inherited disease is Friedrich's Ataxia or Huntington's Disease.

[0128] In some embodiments of the above aspects, the first gRNA further comprises a spacer sequence targeting a region within the amplified trinucleotide repeat sequence or a proximal region of the amplified trinucleotide repeat sequence. In some embodiments, the second gRNA further comprises a spacer sequence targeting a region within the amplified trinucleotide repeat sequence or a proximal region of the amplified trinucleotide repeat sequence.

[0129] In some embodiments of the above aspects, the RGN polypeptide has at least 95% or 100% sequence identity to any one of SEQ ID NOs: 1-20.

[0130] In some embodiments of the above aspects, the gRNA, the first gRNA, the second gRNA, or the first gRNA and the second gRNA are selected from gRNAs selected from the group consisting of: a) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:1; b) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:43, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:2; c) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:3; d) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:45, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:4 has an amino acid sequence with at least 90%, at least 95% or 100% sequence identity; e) a gRNA comprising a CRISPR repeat sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042 and a tracrRNA with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042, wherein the RGN polypeptide has an amino acid sequence with at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:5;f) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 26 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 47 or to nucleotides 24-138 of SEQ ID NO: 1043, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6; g) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6; NO:7 having at least 90%, at least 95% or 100% sequence identity; h) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:50, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8; NO:30 has at least 90%, at least 95%, or 100% sequence identity to a CRISPR repeat sequence and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:10;k) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:31 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:52, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 11; l) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:32 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:53, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 12; m) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:33 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:54, wherein the RGN polypeptide has an amino acid sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:13; n) a gRNA comprising a CRISPR repeat sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:34 and a tracrRNA that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:55, wherein the RGN polypeptide has an amino acid sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:14; o) a gRNA comprising a CRISPR repeat sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:35 and a tracrRNA that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:56, wherein the RGN polypeptide has an amino acid sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:13; NO:15 having an amino acid sequence with at least 90%, at least 95%, or 100% sequence identity; p) a gRNA comprising a CRISPR repeat sequence with at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:36 and a tracrRNA with at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:57, wherein the RGN polypeptide has an amino acid sequence with at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:16;q) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 17; r) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58 or 60, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 18; s) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 18; NO:40 having at least 90%, at least 95% or 100% sequence identity and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:61, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:19; and t) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:62, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:20.;

[0131] In some embodiments of the above aspects, the cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the cell is derived from a dog, cat, mouse, rat, rabbit, horse, cow, pig or human.

[0132] In another aspect, the present disclosure provides a method for producing genetically modified mammalian hematopoietic progenitor cells with reduced expression of BCL11A mRNA and protein, the method comprising introducing into isolated human hematopoietic progenitor cells: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20, or a polynucleotide encoding the RGN polypeptide, wherein the polynucleotide encoding the RGN polypeptide is operably linked to a promoter to enable expression of the RGN polypeptide in the cell; and b) a guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell, whereby the RGN and the gRNA are expressed in the cell and cleaved at the BCL11A enhancer region, resulting in genetic modification of the human hematopoietic progenitor cells and reduced expression of BCL11A mRNA and / or protein.

[0133] In some embodiments of the above aspects, the RGN polypeptide has at least 95% or 100% sequence identity to any one of SEQ ID NOs: 1-20.

[0134] In some embodiments of the above aspects, the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:1; b) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:43, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:2; c) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:23 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:3; d) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:45, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:4; e) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:25 or to SEQ ID a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5;f) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 26 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 47 or to nucleotides 24-138 of SEQ ID NO: 1043, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6; g) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6; NO:7 having at least 90%, at least 95% or 100% sequence identity; h) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:50, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8; NO:30 has at least 90%, at least 95%, or 100% sequence identity to a CRISPR repeat sequence and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:10;k) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:31 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:52, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 11; l) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:32 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:53, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 12; m) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:33 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: NO:54, wherein the RGN polypeptide has an amino acid sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:13; n) a gRNA comprising a CRISPR repeat sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:34 and a tracrRNA that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:55, wherein the RGN polypeptide has an amino acid sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:14; o) a gRNA comprising a CRISPR repeat sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:35 and a tracrRNA that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:56, wherein the RGN polypeptide has an amino acid sequence that has at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:13; NO:15 having an amino acid sequence with at least 90%, at least 95%, or 100% sequence identity; p) a gRNA comprising a CRISPR repeat sequence with at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:36 and a tracrRNA with at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:57, wherein the RGN polypeptide has an amino acid sequence with at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:16;q) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:37 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:58, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:17; r) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:38 and a tracrRNA having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:59 or 60, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:18; s) a gRNA comprising NO:39 or 40 having at least 90%, at least 95% or 100% sequence identity and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:61, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:19; and t) a gRNA comprising a CRISPR repeat sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:62, wherein the RGN polypeptide has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:20.;

[0135] In some embodiments of the above aspects, the gRNA further comprises a spacer sequence targeting a region within the BCL11A enhancer region or a proximal region of the BCL11A enhancer region.

[0136] In another aspect, the present disclosure provides a method of treating a disease, disorder or condition, comprising administering to a subject in need thereof the pharmaceutical composition described above.

[0137] In some embodiments of the above aspects, the disease, disorder, or condition is associated with a causal mutation and the pharmaceutical composition corrects the causal mutation.

[0138] In some embodiments of the above aspects, the subject is at risk for developing the disease, disorder, or condition.

[0139] In another aspect, the disclosure provides use of a nucleic acid molecule, vector, cell, RGN polypeptide, RNP complex or system described above for treating a disease, disorder or condition in a subject in need thereof.

[0140] In some embodiments of the above aspects, the disease, disorder, or condition is associated with a causal mutation and the treatment comprises correcting the causal mutation.

[0141] In some embodiments of the above aspects, the subject is at risk for developing the disease, disorder, or condition.

[0142] In another aspect, the disclosure provides use of a nucleic acid molecule, vector, cell, RGN polypeptide, RNP complex or system as described above in the preparation of a medicament for treating a disease, disorder or condition.

[0143] In some embodiments of the above aspects, the disease is associated with a causal mutation and the drug corrects the causal mutation.

[0144] In another aspect, the present disclosure provides a single guide RNA comprising a nucleic acid molecule containing the crRNA described above and a nucleic acid molecule containing the tracrRNA described above.

[0145] On the other hand, the present disclosure provides a dual-guide RNA comprising a nucleic acid molecule containing the crRNA described above and a nucleic acid molecule containing the tracrRNA described above. DETAILED DESCRIPTION OF THE INVENTION

[0147] Many modifications and other embodiments of the inventions described herein will be considered by those skilled in the art with the benefit of the above description and the teachings of the associated drawings. Therefore, it is to be understood that the invention is not limited to the specific embodiments disclosed, but rather these modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terms are used herein, they are used in a generic and descriptive sense only and not for the purpose of limitation.

[0148] I. Overview

[0149] RNA-guided nucleases (RGNs) allow targeted manipulation of specific sites within the genome and are very useful in the context of gene targeting for therapeutic and research applications. For example, RNA-guided nucleases have been used for genome engineering by stimulating nonhomologous end joining and homologous recombination in various organisms including mammals. The compositions and methods described herein can be used to generate single-stranded or double-stranded breaks in polynucleotides, modify polynucleotides, detect specific sites within polynucleotides, or modify the expression of specific genes.

[0150] RNA-guided nucleases disclosed herein can change gene expression by modifying a target nucleic acid molecule comprising a target sequence. In a specific embodiment, RNA-guided nucleases are directed to a target sequence (e.g., a target DNA sequence) by a guide RNA (gRNA) as part of a clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nuclease system. RGN is considered to be "RNA-guided", because guide RNA forms a complex with RNA-guided nucleases to guide RNA-guided nucleases to bind to a target sequence, and in some embodiments, single-strand or double-strand breaks are introduced at a target sequence (e.g., a target DNA sequence). After the target sequence is cut, the break can be repaired so as to modify the sequence of the target nucleic acid molecule during the repair process. Therefore, a method for modifying a target nucleic acid molecule in a host cell using an RNA-guided nuclease is provided herein. For example, RNA-guided nucleases can be used to modify a target sequence at a eukaryotic or prokaryotic genomic site.

[0151] II. RNA-guided nucleases

[0152] RNA-guided nucleases are provided herein. The term RNA-guided nuclease (RGN) refers to a polypeptide that binds to a specific target sequence (e.g., a target DNA sequence) in a sequence-specific manner and is directed to a target sequence by a guide RNA molecule that is complexed with the polypeptide and hybridized with the target sequence. Although RNA-guided nucleases can cut the target sequence when bound, the term RNA-guided nucleases also include RNA-guided nucleases that can bind but not cut the target sequence. RNA-guided nucleases that cut the target sequence can cause single-stranded or double-stranded breaks. RNA-guided nucleases that can only cut the single strand of a double-stranded target nucleic acid molecule are referred to herein as nickases.

[0153] The RNA-guided nucleases disclosed herein include LPG10165, LPG10166, LPG10167, LPG10168, LPG10169, LPG10171, LPG10186, LPG10190, LPG10191, LPG10194, LPG10195, LPG10196, LPG10197, LPG10198, LPG10200, LPG10203, LPG10204, LPG10205, LPG10207 and LPG10208 RNA-guided nucleases, whose amino acid sequences are represented by SEQ ID NOs: 1-20, respectively, as well as active fragments or variants thereof, which retain the ability to bind to a target sequence in an RNA-guided, sequence-specific manner. In some of these embodiments, the active fragments or variants of LPG10165, LPG10166, LPG10167, LPG10168, LPG10169, LPG10171, LPG10186, LPG10190, LPG10191, LPG10194, LPG10195, LPG10196, LPG10197, LPG10198, LPG10200, LPG10203, LPG10204, LPG10205, LPG10207 or LPG10208 RGN are capable of cleaving a single-stranded or double-stranded target sequence. In some embodiments, an active variant of LPG10165, LPG10166, LPG10167, LPG10168, LPG10169, LPG10171, LPG10186, LPG10190, LPG10191, LPG10194, LPG10195, LPG10196, LPG10197, LPG10198, LPG10200, LPG10203, LPG10204, LPG10205, LPG10207, or LPG10208 RGN comprises the sequence of SEQ ID any one of NO:1-20 having an amino acid sequence of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0154] In some specific embodiments, the active variant of LPG10165RGN comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10166RGN comprises an amino acid sequence having at least 83% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10167RGN comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10168RGN comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10169RGN comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10171RGN comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence shown in SEQ ID NO:6, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10186RGN comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10190RGN comprises an amino acid sequence having at least 84% sequence identity with the amino acid sequence shown in SEQ ID NO:8, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10191RGN comprises an amino acid sequence having at least 73% sequence identity with the amino acid sequence shown in SEQ ID NO:9, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10194RGN comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:10, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10195RGN comprises an amino acid sequence having at least 88% sequence identity to the amino acid sequence shown in SEQ ID NO: 11, and retains RNA-guided sequence-specific binding activity.In some embodiments, the active variant of LPG10196RGN comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence shown in SEQ ID NO: 12, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10197RGN comprises an amino acid sequence having at least 92% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10198RGN comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence shown in SEQ ID NO: 14, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10200RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence shown in SEQ ID NO: 15, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10203RGN comprises an amino acid sequence having at least 78% sequence identity to the amino acid sequence shown in SEQ ID NO: 16, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LEPG10204RGN comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence shown in SEQ ID NO: 17, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10205RGN comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10207RGN comprises an amino acid sequence having at least 88% sequence identity to the amino acid sequence shown in SEQ ID NO: 19, and retains RNA-guided sequence-specific binding activity. In some embodiments, the active variant of LPG10208RGN comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence shown in SEQ ID NO:20, and retains RNA-guided sequence-specific binding activity.

[0155] In certain embodiments, the active fragment of LPG10165, LPG10166, LPG10167, LPG10168, LPG10169, LPG10171, LPG10186, LPG10190, LPG10191, LPG10194, LPG10195, LPG10196, LPG10197, LPG10198, LPG10200, LPG10203, LPG10204, LPG10205, LPG10207, or LPG10208 RGN comprises SEQ ID At least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300 or more consecutive amino acid residues of the amino acid sequence shown in any one of NO: 1-20. The RNA-guided nuclease provided herein can include at least one nuclease domain (e.g., DNA enzyme, RNA enzyme domain) and at least one RNA recognition and / or RNA binding domain to interact with the guide RNA. Other domains that may be present in the RNA-guided nuclease provided herein include, but are not limited to, a DNA binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain. In specific embodiments, the RNA-guided nucleases provided herein can comprise a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to one or more of a DNA binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain.

[0156] The target sequence is combined with the RNA-guided nuclease provided herein. In the case where the target sequence is double-stranded (e.g., double-stranded DNA), the non-target strand of the target sequence is hybridized with the guide RNA associated with the RNA-guided nuclease. If the polypeptide has nuclease activity, the target strand and / or the non-target strand of the target sequence (e.g., target DNA sequence) can be subsequently cut by the RNA-guided nuclease. The term "cutting" or "cleavage" refers to the hydrolysis of at least one phosphodiester bond in the main chain of one or both chains of a double-stranded target sequence (e.g., target DNA sequence), which can result in single-strand or double-strand breaks in the target sequence. The RGN disclosed herein can cut nucleotides in a polynucleotide, acting as an endonuclease, or can be an exonuclease, removing continuous nucleotides from the ends (5' and / or 3' ends) of the polynucleotide. In other embodiments, the disclosed RGN can cut nucleotides of the target polynucleotide in any position of the polynucleotide, thereby acting as an endonuclease and an exonuclease at the same time. The RGN disclosed herein cuts the target polynucleotide and can result in staggered breaks or flat ends.

[0157] The RNA-guided nucleases of the present disclosure may be wild-type sequences derived from bacterial or archaeal species. Alternatively, the RNA-guided nucleases may be variants or fragments of wild-type polypeptides. For example, wild-type RGNs may be modified to alter nuclease activity or to alter PAM specificity. In some embodiments, the RNA-guided nucleases are not naturally occurring.

[0158] In certain embodiments, the RNA-guided nuclease acts as a nickase, cutting only the single strand of a double-stranded target sequence (e.g., a target DNA sequence). This RNA-guided nuclease has a single functional nuclease domain. In a specific embodiment, the nickase is capable of cutting the target strand or non-target strand of a double-stranded target sequence (e.g., a target DNA sequence). In some of these embodiments, other nuclease domains have been mutated so that the nuclease activity is reduced or eliminated. In an embodiment using a nickase, in order to achieve double-stranded cleavage of a double-stranded target sequence (e.g., a target DNA sequence), two nickases are required, each nickase cutting a single strand in the double-stranded target sequence.

[0159] In other embodiments, the RNA-guided nucleases all lack nuclease activity, referred to herein as nuclease-inactive or nuclease-inactive. Any method known in the art for introducing mutations into an amino acid sequence, such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used to generate nickase- or nuclease-inactive RGNs. See, e.g., U.S. Patent Publication No. 2014 / 0068797 and U.S. Patent No. 9,790,490; each of which is incorporated by reference in its entirety.

[0160] RNA-guided nucleases lacking nuclease activity can be used to deliver fusion polypeptides, polynucleotides, or small molecule payloads to specific genomic locations. In some of these embodiments, the RGN polypeptide or guide RNA can be fused to a detectable tag to allow detection of a specific sequence. As a non-limiting example, a nuclease-inactive RGN can be fused to a detectable tag (e.g., a fluorescent protein) and targeted to a specific sequence associated with a disease to allow detection of a disease-associated sequence.

[0161] Alternatively, nuclease-inactivated RGNs can target specific genomic locations to alter the expression of a desired gene (i.e., a target gene). In some embodiments, the binding of a nuclease-inactivated RNA-guided nuclease to a target sequence results in reduced expression of the target gene by interfering with the binding of an RNA polymerase or transcription factor in the targeted genomic region. In other embodiments, the RGN (e.g., a nuclease-inactivated RGN) or its composite guide RNA further comprises an expression regulator that, when bound to a target sequence in the target gene, is used to inhibit or activate the expression of the target gene. In some of these embodiments, the expression regulator regulates the expression of the target gene by an epigenetic mechanism.

[0162] In other embodiments, a nuclease-inactivated RGN or an RGN with nickase activity can be targeted to a specific genomic location to modify the sequence of a target polynucleotide by fusion with a base editing polypeptide, such as a deaminase polypeptide or an active variant thereof or a fragment thereof, which directly chemically modifies (e.g., deaminates) a nucleobase, resulting in conversion from one nucleobase to another. The base editing polypeptide can be fused to the RGN at its N-terminus or C-terminus. In addition, the base editing polypeptide can be fused to the RGN via a peptide linker. Non-limiting examples of deaminase polypeptides that can be used in such compositions and methods include cytosine deaminase or adenine deaminase (such as the adenine deaminase base editors described in Gaudelli et al., (2017) Nature 551:464-471, U.S. Patent Publication Nos. 2017 / 0121693 and 2018 / 0073012, and International Patent Publication No. WO 2018 / 027078, or International Patent Publication Nos. WO 2020 / 139783 and WO 2022 / 056254, and any of the deaminases disclosed in International Patent Application No. PCT / US2022 / 021271 filed on March 22, 2022, each of which is incorporated herein by reference in its entirety). In one embodiment, the deaminase polypeptide useful in such compositions and methods is a cytosine deaminase or adenine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 481-552. In one embodiment, the deaminase polypeptide useful in such compositions and methods is a cytosine deaminase or adenine deaminase having a sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any one of the amino acid sequences shown in SEQ ID NOs: 481-552. In some embodiments, the deaminase polypeptide useful for the compositions and methods of the present disclosure is a deaminase disclosed in Table 17 of International Patent Publication No. WO 2020 / 139783, which is incorporated herein by reference in its entirety. In addition, it is known in the art that certain fusion proteins between RGN and base editing enzymes (e.g., cytosine deaminases) can also include at least one uracil stabilizing polypeptide that increases the mutation rate of cytosine, deoxycytosine, or cytosine to thymidine, deoxythymidine, or thymine in nucleic acid molecules by deaminases.Non-limiting examples of uracil stabilizing polypeptides include those disclosed in International Patent Publication No. WO 2021 / 217002, which is incorporated herein by reference in its entirety, including USP2 (SEQ ID NO: 564) and uracil glycosylase inhibitor (UGI) domain (SEQ ID NO: 565), which can improve base editing efficiency. Therefore, the fusion protein may include RGN described herein or a variant thereof, a deaminase, and optionally at least one uracil stabilizing polypeptide, such as UGI or USP2. In certain embodiments, the RGN fused to the base editing polypeptide is a nicking enzyme that cuts a DNA strand that is not acted upon by a base editing polypeptide (e.g., a deaminase).

[0163] RNA-guided nucleases fused to polypeptides or domains can be separated or connected by joints. The term "joint" used herein refers to a chemical group or molecule connecting two molecules or parts, for example, a binding domain and a cleavage domain of a nuclease. In some embodiments, the joint connects the gRNA binding domain of the RNA-guided nuclease and a base editing polypeptide, such as a deaminase. In some embodiments, the joint connects the RGN and deaminase in which the nuclease is inactivated. Typically, the joint is located between or on both sides of two groups, molecules or other parts, and is covalently bonded to each group, molecule or other part, thereby connecting the two. In some embodiments, the joint is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the joint is an organic molecule, a group, a polymer or a chemical part. In some embodiments, the length of the linker is 5-100 amino acids, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0164] The RNA-guided nucleases of the present disclosure may comprise at least one nuclear localization signal (NLS) to enhance the transport of the RGN to the nucleus. Nuclear localization signals are known in the art and typically comprise a stretch of basic amino acids (see, e.g., Lange et al., J. Biol. Chem. (2007) 282: 5101-5105). In some embodiments, the RGN comprises 2, 3, 4, 5, 6 or more nuclear localization signals. The nuclear localization signal may be a heterologous NLS. Non-limiting examples of nuclear localization signals useful for the RGNs of the present disclosure are the nuclear localization signals of SV40 large T antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al., (2015) Bioconjug Chem 26 (6): 1004-7). In specific embodiments, the RGN comprises an NLS sequence as shown in SEQ ID NO: 168 or 170. The RGN may comprise one or more NLS sequences at its N-terminus, C-terminus, or both the N-terminus and the C-terminus. For example, an RGN may contain two NLS sequences at the N-terminal region and four NLS sequences at the C-terminal region.

[0165] Other localization signal sequences known in the art to localize polypeptides to specific subcellular locations can also be used to target RGNs, including but not limited to plastid localization sequences, mitochondrial localization sequences, and dual targeting signal sequences that target plastids and mitochondria (see, e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physioldx.doi.org / 10.3389 / fphys.2015.00259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soll (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al., (2009) FEBS J 276: 1187-1195; Silva-Filho (2003) Curr Opin Plant Biol 6: 589-595; Peeters and Small (2001) Biochim Biophys Acta 1541: 54-63; Murcha et al. (2014) J Exp Bot 65: 6301-6335; Mackenzie (2005) Trends Cell Biol 15: 548-554; Glaser et al. (1998) Plant Mol Biol 38: 311-338).

[0166] In certain embodiments, the RNA-guided nuclease of the present disclosure comprises at least one cell-penetrating domain that promotes cellular uptake of RGN. Cell-penetrating domains are known in the art and typically include a segment of positively charged amino acid residues (i.e., polycationic cell-penetrating domains), alternating polar amino acid residues and non-polar amino acid residues (i.e., amphipathic cell-penetrating domains) or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (see, e.g., Milletti F. (2012) Drug Discov Today 17: 850-860). A non-limiting example of a cell-penetrating domain is a transactivating transcriptional activator (TAT) from human immunodeficiency virus 1.

[0167] The nuclear localization signal, plastid localization signal, mitochondrial localization signal, dual targeting localization signal and / or cell penetration domain can be located at the amino terminus (N-terminus), carboxyl terminus (C-terminus), or an internal position of the RNA-guided nuclease.

[0168] The RGNs of the present disclosure can be fused directly or indirectly to an effector domain, such as a cleavage domain, a deaminase domain, or an expression regulator domain, via a linker peptide. Such domains can be located at the N-terminus, C-terminus, or internal position of the RNA-guided nuclease. In some of these embodiments, the RGN component of the fusion protein is a nuclease-inactivated RGN or a nickase.

[0169] In some embodiments, the RGN fusion protein comprises a cleavage domain, which is any domain capable of cleaving a polynucleotide (i.e., RNA, DNA, or RNA / DNA hybrid) and includes, but is not limited to, restriction endonucleases and homing endonucleases, such as Type IIS endonucleases (e.g., FokI) (see, e.g., Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388; Linn et al., (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).

[0170] In other embodiments, the RGN fusion protein comprises a deaminase domain that deaminates nucleobases, resulting in conversion from one nucleobase to another, and includes, but is not limited to, a cytosine deaminase or an adenine deaminase (see, e.g., Gaudelli et al., (2017) Nature 551:464-471, U.S. Patent Publication Nos. 2017 / 0121693 and 2018 / 0073012, and International Patent Publication No. WO 2018 / 027078, or International Patent Publication No. WO 2020 / 139783 and WO2022 / 056254 and any deaminase disclosed in International Application No. PCT / US2022 / 021271 filed on March 22, 2022, each of which is incorporated herein by reference in its entirety. In some embodiments, the effector domain of the RGN fusion protein can be an expression regulator domain that acts to upregulate or downregulate transcription. The expression regulator domain can be an epigenetic modification domain, a transcription repression domain, or a transcription activation domain.

[0171] In some embodiments, the expression regulator of RGN fusion protein comprises an epigenetic modification domain, which covalently modifies DNA or histone to change histone structure and / or chromosome structure without changing the DNA sequence, resulting in changes in gene expression (i.e., upregulation or downregulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and SUMOylation of histones, as well as methylation and hydroxymethylation of cytosine residues in DNA. Non-limiting examples of epigenetic modification domains include histone acetyltransferase domains, histone deacetylase domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.

[0172] In other embodiments, the expression regulator of the fusion protein includes a transcriptional repressor domain that interacts with a transcriptional control element and / or a transcriptional regulatory protein (such as an RNA polymerase and a transcription factor) to reduce or terminate the transcription of at least one gene. Transcriptional repressor domains are known in the art and include, but are not limited to, Sp1-like repressors, IκB, and Krüppel-associated box (KRAB) domains.

[0173] Also in other embodiments, the expression regulator of fusion protein comprises a transcriptional activation domain, which interacts with a transcriptional control element and / or a transcriptional regulatory protein (such as an RNA polymerase and a transcription factor) to increase or activate the transcription of at least one gene. Transcriptional activation domains are known in the art, including but not limited to, herpes simplex virus VP16 activation domain and NFAT activation domain.

[0174] The RGN polypeptides of the present disclosure may include a detectable label or purification tag. The detectable label or purification tag may be located directly or indirectly at the N-terminus, C-terminus, or internal position of the RNA-guided nuclease via a linker peptide. In some of these embodiments, the RGN component of the fusion protein is a nuclease-inactive RGN. In other embodiments, the RGN component of the fusion protein is a RGN with nickase activity.

[0175] A detectable label is a molecule that can be visualized or otherwise observed. A detectable label can be fused to an RGN as a fusion protein (e.g., a fluorescent protein) or can be a small molecule conjugated to an RGN polypeptide that can be detected by the naked eye or otherwise. Detectable labels that can be fused to an RGN of the present disclosure as a fusion protein include any detectable protein domain, including, but not limited to, fluorescent proteins or protein domains that can be detected with specific antibodies. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreen1) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellow1). Non-limiting examples of small molecule detectable labels include radioactive labels such as 3 H and 35 S.

[0176] The RGN polypeptide may also include a purification tag, which is any molecule that can be used to separate a protein or fusion protein from a mixture (e.g., a biological sample, culture medium). Non-limiting examples of purification tags include biotin, myc, maltose binding protein (MBP), glutathione-S-transferase (GST), and 3X FLAG tags.

[0177] III. Guide RNA

[0178] The present disclosure provides guide RNAs that target the associated RGN to a target sequence and polynucleotides encoding the guide RNAs. The term "guide RNA" refers to a nucleotide sequence that has sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and direct the associated RNA-guided nuclease to bind specifically to the sequence of the target nucleotide sequence. More specifically, when the target nucleotide sequence is double-stranded like DNA, the target nucleotide sequence consists of a target strand (including a PAM sequence) and a non-target strand. In these embodiments, the guide RNA has sufficient complementarity with the non-target strand of the double-stranded target sequence (e.g., a target DNA sequence) so that the guide RNA hybridizes with the non-target strand and directs the associated RNA-guided nuclease (RGN) to bind specifically to the target sequence (e.g., a target DNA sequence). Therefore, in some embodiments, the guide RNA includes a spacer sequence identical to the sequence of the target strand, except that uracil (U) replaces thymidine (T) in the guide RNA.

[0179] The guide RNA of each RGN is one or more RNA molecules (usually one or more) that can bind to the RGN and guide the RGN to bind to a specific target sequence, and in those embodiments where the RGN has nickase or nuclease activity, also cuts the target strand and / or non-target strand. Typically, guide RNAs include CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), although some RGNs do not require tracrRNA. Natural guide RNAs containing both crRNA and tracrRNA typically contain two independent RNA molecules that hybridize to each other through the repeat sequence of crRNA and the anti-repeat sequence of tracrRNA.

[0180] The present invention provides CRISPR RNA (crRNA) or polynucleotides encoding CRISPR RNA, which together with tracrRNA target the associated RGN to the target sequence. CrRNA comprises a spacer sequence and a CRISPR repeat sequence. A "spacer sequence" has a nucleotide sequence that directly hybridizes with a non-target strand of a target sequence of interest (e.g., a target DNA sequence). The spacer sequence is modified to have complete or partial complementarity with the non-target strand of the target sequence of interest. In some embodiments, the spacer sequence may include from about 8 nucleotides to about 30 nucleotides, or more. For example, the length of the spacer sequence may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In some embodiments, the length of the spacer sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. In some embodiments, the length of the spacer sequence is about 10 to about 26 nucleotides, or about 12 to about 30 nucleotides. In some embodiments, when optimally aligned using an appropriate alignment algorithm, the degree of complementarity between the spacer sequence and the non-target strand of the target sequence (e.g., target DNA sequence) is between 50% and 99% or more, including but not limited to about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the spacer sequence and the non-target strand of the target sequence (e.g., target DNA sequence) is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the spacer sequence may be identical in sequence to the target strand of the target sequence. In some of those embodiments in which the target sequence is a target DNA sequence, the spacer sequence may be identical in sequence to the target strand of the target DNA sequence, except that the thymidine (T) in the target strand is replaced by the uracil (U) in the spacer sequence.In specific embodiments, the spacer sequence has no secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9:133-148) and RNAfold (see, e.g., Gruber et al., (2008) Cell 106(1):23-24).

[0181] The crRNA of the present disclosure comprises a spacer sequence capable of targeting a bound RGN polypeptide to a target DNA sequence, wherein the target strand of the target DNA sequence has a nucleotide sequence shown in any one of SEQ ID NOs: 344-464, 573-641, 667-677, 684-747, 770-817, 826-1039 and 1046-1057.

[0182] Together with the spacer sequence, crRNA also includes CRISPR RNA repeats. CRISPR RNA repeats include nucleotide sequences that themselves or in conjunction with hybridized tracrRNA form a structure recognized by RGN molecules. In various embodiments, CRISPR RNA repeats may include about 8 nucleotides to about 30 nucleotides, or more. For example, the length of the CRISPR repeats may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In specific embodiments, the CRISPR repeat sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence is about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more when optimally aligned using a suitable alignment algorithm. In specific embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0183] In specific embodiments, the CRISPR repeat sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 21-41, or nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045, or an active variant or fragment thereof, which, when contained in a guide RNA, is capable of directing sequence-specific binding of an associated RNA-guided nuclease provided herein to a target sequence of interest. In certain embodiments, an active CRISPR repeat sequence variant of a wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs:21-41, or nucleotides 1-17 of SEQ ID NOs:1041 or 1042, or to nucleotides 1-22 of SEQ ID NOs:1044 or 1045. In certain embodiments, the active CRISPR repeat sequence fragment of the wild-type sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs:21-41, or nucleotides 1-17 of SEQ ID NOs:1041 or 1042, or nucleotides 1-22 of SEQ ID NOs:1044 or 1045.

[0184] In certain embodiments, the crRNA is not naturally occurring. In some of these embodiments, the specific CRISPR repeat sequence is unrelated to an engineered spacer sequence in nature, and the CRISPR repeat sequence is considered to be heterologous to the spacer sequence. In certain embodiments, the spacer sequence is a non-naturally occurring engineered sequence.

[0185] The guide RNA disclosed herein includes crRNA and trans-activation CRISPR RNA (tracrRNA). The tracrRNA molecule includes a nucleotide sequence, which includes a region having sufficient complementarity to hybridize with the CRISPR repeat sequence of crRNA, referred to herein as an anti-repeat sequence. In some embodiments, the tracrRNA molecule also includes a region with a secondary structure (e.g., a stem loop) or forms a secondary structure when hybridized with its corresponding crRNA. In a specific embodiment, the region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is located at the 5' end of the molecule, and the 3' end of the tracrRNA includes a secondary structure. The region of this secondary structure generally includes several hairpin structures, including a nexus hairpin adjacent to the anti-repeat sequence. The nexus forms the core of the interaction between the guide RNA and the RGN, located at the intersection between the guide RNA, the RGN and the target DNA. The nexus hairpin generally has a conserved nucleotide sequence at the base of the hairpin stem, and the motif UNANNC (SEQ ID NO: 566) is found in many nexus hairpins of tracrRNA. In an embodiment, the tracrRNA used in the guide RNA or RGN system of the present disclosure comprises a non-classical sequence at the base of the hairpin stem to which the hairpin is attached, including UNANNG (SEQ ID NO: 567), CNANNC (SEQ ID NO: 568), CNANNU (SEQ ID NO: 569), UNANNU (SEQ ID NO: 570), CNANNG (SEQ ID NO: 571) and CNCNNU (SEQ ID NO: 572). There is usually a terminal hairpin at the 3' end of the tracrRNA, which may vary in structure and number, but generally includes a GC-rich Rho-independent transcription terminator hairpin followed by a run of Us at the 3' end. See, e.g., Briner et al., (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc; doi: 10.1101 / pdb.top090902, and U.S. Patent Publication No. 2017 / 0275648, each of which is incorporated herein by reference in its entirety.

[0186] In various embodiments, the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence includes about 8 nucleotides to about 30 nucleotides or more. For example, the length of the base pairing region between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In specific embodiments, the base-paired region between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence is about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more when optimally aligned using a suitable alignment algorithm. In certain embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0187] In various embodiments, the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides. For example, the length of the tracrRNA can be about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210 or more nucleotides. In specific embodiments, the tracrRNA is 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210 nucleotides or more in length. In certain embodiments, the tracrRNA is about 57 to about 115 nucleotides in length, including about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105 In the present invention, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, and about 115 nucleotides.In a specific embodiment, the length of the tracrRNA is 59 to 115 nucleotides, including 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 108, 109, 110, 111, 112, 113, 114, and 115 nucleotides.

[0188] In a specific embodiment, the tracrRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NO: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045, or an active variant or fragment thereof, which, when contained in a guide RNA, is capable of directing sequence-specific binding of an associated RNA-guided nuclease provided herein to a target DNA sequence of interest. In certain embodiments, the active tracrRNA sequence variants include nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the nucleotide sequences set forth in SEQ ID NOs:42-62, nucleotides 19-111 of SEQ ID NO:1040, nucleotides 22-85 of SEQ ID NOs:1041 or 1042, nucleotides 24-138 of SEQ ID NO:143, nucleotides 27-96 of SEQ ID NO:1044, or nucleotides 27-95 of SEQ ID NO:1045. In certain embodiments, the active tracrRNA sequence fragment comprises any one of the nucleotide sequences set forth in SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NOs: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of nucleotides 27-95 of SEQ ID NO: 1045.

[0189] When two polynucleotide sequences hybridize to each other under stringent conditions, the two polynucleotide sequences can be considered to be substantially complementary. Similarly, if the guide RNA bound to the RGN binds to the target sequence under stringent conditions, the RGN is considered to bind to a specific target sequence in a sequence-specific manner. "Stringent conditions" or "stringent hybridization conditions" refer to the expected conditions under which two polynucleotide sequences hybridize to each other, and the degree of detection is higher than other sequences (e.g., at least 2 times the background). Stringent conditions depend on the sequence and will vary in different situations. Generally, stringent conditions are salt concentrations less than about 1.5M Na ions, typically about 0.01 to 1.0M Na ion concentration (or other salts) at pH 7.0 to 8.3, and for short sequences (e.g., 10 to 50 nucleotides), the temperature is at least about 30°C, and for long sequences (e.g., greater than 50 nucleotides), the temperature is at least about 60°C. Stringent conditions can also be achieved by adding destabilizing agents (such as formamide). Exemplary low stringency conditions include hybridization with a buffer solution of 30% to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and washing in 1X to 2X SSC (20X SSC=3.0M NaCl / 0.3M trisodium citrate) at 50°C to 55°C. Exemplary moderate stringency conditions include hybridization in 40% to 45% formamide, 1.0M NaCl, 1% SDS at 37°C, and washing in 0.5X-1X SSC at 55-60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C, and washing in 0.1X SSC at 60-65°C. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours. The duration of the wash time is at least sufficient to reach equilibrium.

[0190] Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to the perfectly matched sequence. For DNA-DNA hybrids, Tm can be estimated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138: 267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L; where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Typically, stringent conditions are selected to be about 5°C lower than the thermodynamic melting point (Tm) of the specific sequence and its complement at a defined ionic strength and pH. However, under stringent conditions, hybridization and / or washing can be performed at 1, 2, 3 or 4° C. below the thermodynamic melting point (Tm); under moderately stringent conditions, hybridization and / or washing can be performed at 6, 7, 8, 9 or 10° C. below the thermodynamic melting point (Tm); and under low stringency conditions, hybridization and / or washing can be performed at 11, 12, 13, 14, 15 or 20° C. below the thermodynamic melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, one of ordinary skill will understand that various variations of the stringency of hybridization and / or wash solutions are inherently described. Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds., (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York) provide detailed guidelines for nucleic acid hybridization. See Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (Second Edition, Cold Spring Harbor Laboratory Press, Plainview, New York).

[0191] The term "sequence specific" may also refer to the fact that the RGN polypeptide binds to the target sequence more frequently than to random background sequences.

[0192] Guide RNA can be single guide RNA (sgRNA) or double guide RNA.Single guide RNA includes crRNA and tracrRNA on a single RNA molecule, while double guide RNA includes crRNA and tracrRNA present on two different RNA molecules, hybridized with each other by at least a portion of CRISPR repeats of crRNA and at least a portion of tracrRNA (i.e., anti-repeats), and it may be completely or partially complementary to the CRISPR repeats of crRNA.In some of those embodiments in which guide RNA is a single guide RNA, crRNA and tracrRNA are separated by a joint nucleotide sequence.Generally, the joint nucleotide sequence is a sequence that does not include complementary bases, to avoid forming a secondary structure in the nucleotides of the joint nucleotide sequence or a secondary structure of the nucleotides including the joint nucleotide sequence.In some embodiments, the length of the joint nucleotide sequence between crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or more nucleotides.In a specific embodiment, the length of the joint nucleotide sequence of a single guide RNA is at least 4 nucleotides. In certain embodiments, the linker nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:84.

[0193] Single guide RNA or dual guide RNA can be chemically synthesized or in vitro transcribed. Assays for determining sequence-specific binding between RGN and guide RNA are known in the art, including but not limited to in vitro binding assays between expressed RGN and guide RNA, which can be labeled with a detectable label (e.g., biotin) and used in a sedimentation assay, in which the guide RNA: RGN complex is captured by the detectable label (e.g., using streptavidin beads). Control guide RNAs with unrelated sequences or structures to the guide RNA can be used as negative controls for non-specific binding of RGN to RNA. In certain embodiments, the guide RNA has a backbone sequence that is any one of SEQ ID NOs: 63-83, 1040, 1041, 1042, 1043, 1044, or 1045.

[0194] In certain embodiments, guide RNA can be introduced into target cells, organelles or embryos as RNA molecules.Guide RNA can be transcribed in vitro or chemically synthesized.In other embodiments, the nucleotide sequence encoding guide RNA is introduced into cells or embryos.In some of these embodiments, the nucleotide sequence encoding guide RNA is operably connected to a promoter (e.g., RNA polymerase III promoter).Promoter can be a natural promoter or a heterologous promoter of the guide RNA encoding nucleotide sequence.

[0195] In various embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, in which the guide RNA is associated with an RNA-guided nuclease polypeptide.

[0196] Guide RNA guides the associated RGN to the target nucleotide sequence of interest by hybridizing the guide RNA with the target sequence of interest. The target sequence can be bound (cut in some embodiments) by an RNA-guided nuclease in vitro or in a cell. The target sequence is located in a target polynucleotide and can contain DNA, RNA, or a combination of both, and can be single-stranded or double-stranded. The target sequence can be genomic DNA (i.e., chromosomal DNA), plasmid DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). In those embodiments in which the target sequence is a chromosomal sequence, the chromosomal sequence can be a nuclear, plastid, or mitochondrial chromosomal sequence. In the compositions and methods of the present disclosure, the target sequence is located in a double-stranded target nucleic acid molecule (e.g., a target DNA sequence). In an embodiment, the target sequence is unique in the target genome.

[0197] The target sequence is adjacent to the protospacer adjacent motif (PAM), and the target strand of the target sequence is the strand containing the PAM. The PAM is adjacent to the target sequence and generally contains multiple Ns, where N represents any nucleotide. In some embodiments, the protospacer adjacent motif contains about 1 to about 10 Ns, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides. In specific embodiments, the PAM contains 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns. The PAM can be 5' or 3' of the target sequence on its target strand. The PAM of the RGN disclosed herein is the 3' end of the target sequence on its target strand. Typically, the PAM is a consensus sequence of about 3-4 nucleotides, but in specific embodiments, its length can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides. In various embodiments, the PAM sequence recognized by the RGN of the present disclosure includes the consensus sequence shown in any one of SEQ ID NOs: 127-147. In some embodiments of the above aspects, the crRNA is capable of binding to an RGN polypeptide that is capable of recognizing all protospacer adjacent motifs (PAMs) having a nucleotide sequence shown in any one of SEQ ID NOs: 127-147.

[0198] In certain embodiments, the RNA-guided nuclease having any one of SEQ ID NOs: 1-20 or an active variant or fragment thereof binds to a target nucleotide sequence adjacent to a PAM sequence as set forth in any one of SEQ ID NOs: 127-147. In some embodiments, the RGN is combined with a guide RNA comprising a CRISPR repeat sequence and a tracrRNA, wherein the CRISPR repeat sequence has a nucleotide sequence as shown in any one of SEQ ID NOs: 21-41, or nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045, or an active variant or fragment thereof, and the tracrRNA has a nucleotide sequence as shown in any one of SEQ ID NOs: 42-62, or nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NOs: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045, or an active variant or fragment thereof. The RGN system is further described in Examples 1-3 and Tables 1 and 2 of the specification.

[0199] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 1 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 127 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 21 or an active variant or fragment thereof and a tracrRNA sequence as shown in SEQ ID NO: 42 or an active variant or fragment thereof.

[0200] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:22, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:43, or an active variant or fragment thereof, the RNA-guided nuclease or an active variant or fragment thereof having SEQ ID NO:2 binds to a target nucleotide sequence adjacent to a PAM sequence as shown in any one of SEQ ID NOs:128-131.

[0201] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO:3 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO:132 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:23, or an active variant or fragment thereof, and a tracrRNA sequence of nucleotides 19-111 as shown in SEQ ID NO:44 or SEQ ID NO:1040, or an active variant or fragment thereof.

[0202] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:24, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:45, or an active variant or fragment thereof, the RNA-guided nuclease or an active variant or fragment thereof having SEQ ID NO:4 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO:132.

[0203] In some embodiments, the RNA-guided nuclease having SEQ ID NO:5 or an active variant or fragment thereof binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:133 when bound to a guide RNA comprising a CRISPR repeat sequence of nucleotides 1-17 as shown in SEQ ID NO:25 or SEQ ID NO:1041 or 1042, or an active variant or fragment thereof and a tracrRNA sequence of nucleotides 22-85 as shown in SEQ ID NO:46 or SEQ ID NO:1041 or 1042, or an active variant or fragment thereof.

[0204] In some embodiments, the RNA-guided nuclease having SEQ ID NO:6 or an active variant or fragment thereof binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO:134 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:26, or an active variant or fragment thereof, and a tracrRNA sequence of nucleotides 24-138 as shown in SEQ ID NO:47 or SEQ ID NO:1043, or an active variant or fragment thereof.

[0205] In some embodiments, the RNA-guided nuclease having SEQ ID NO:7, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as set forth in SEQ ID NO:135 when bound to a guide RNA comprising a CRISPR repeat sequence of nucleotides 1-22 as set forth in SEQ ID NO:27 or SEQ ID NO:1044 or 1045, or an active variant or fragment thereof and a tracrRNA sequence of nucleotides 27-96 as set forth in SEQ ID NO:48 or SEQ ID NO:1044, or nucleotides 27-95 of SEQ ID NO:1045, or an active variant or fragment thereof.

[0206] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:28, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:49, or an active variant or fragment thereof, the RNA-guided nuclease or an active variant or fragment thereof having SEQ ID NO:8 binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:136 or 137.

[0207] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:29, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:50, or an active variant or fragment thereof, the RNA-guided nuclease having SEQ ID NO:9, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO:138.

[0208] In some embodiments, the RNA-guided nuclease or its active variant or fragment having SEQ ID NO: 10 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 139 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 30 or an active variant or fragment thereof and a tracrRNA sequence as shown in SEQ ID NO: 51 or an active variant or fragment thereof.

[0209] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 11 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 140 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 31 or an active variant or fragment thereof and a tracrRNA sequence as shown in SEQ ID NO: 52 or an active variant or fragment thereof.

[0210] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 12 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 141 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 32, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO: 53, or an active variant or fragment thereof.

[0211] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 13 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 142 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 33 or an active variant or fragment thereof and a tracrRNA sequence as shown in SEQ ID NO: 54 or an active variant or fragment thereof.

[0212] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 14 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 143 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 34 or an active variant or fragment thereof and a tracrRNA sequence as shown in SEQ ID NO: 55 or an active variant or fragment thereof.

[0213] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 15 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 144 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 35 or an active variant or fragment thereof and a tracrRNA sequence as shown in SEQ ID NO: 56 or an active variant or fragment thereof.

[0214] In some embodiments, the RNA-guided nuclease or its active variant or fragment having SEQ ID NO: 16 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 145 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 36, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO: 57, or an active variant or fragment thereof.

[0215] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 17 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 146 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 37 or an active variant or fragment thereof and a tracrRNA sequence as shown in SEQ ID NO: 58 or an active variant or fragment thereof.

[0216] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as set forth in SEQ ID NO:38, or an active variant or fragment thereof, and a tracrRNA sequence as set forth in SEQ ID NO:59, or an active variant or fragment thereof, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 18 binds to a target nucleotide sequence adjacent to a PAM sequence as set forth in SEQ ID NO: 146. In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as set forth in SEQ ID NO:39, or an active variant or fragment thereof, and a tracrRNA sequence as set forth in SEQ ID NO:60, or an active variant or fragment thereof, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 18 binds to a target nucleotide sequence adjacent to a PAM sequence as set forth in SEQ ID NO: 146.

[0217] In some embodiments, the RNA-guided nuclease or its active variant or fragment thereof having SEQ ID NO: 19 binds to a target nucleotide sequence adjacent to the PAM sequence as shown in SEQ ID NO: 132 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO: 40, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO: 61, or an active variant or fragment thereof.

[0218] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as set forth in SEQ ID NO: 41, or an active variant or fragment thereof, and a tracrRNA sequence as set forth in SEQ ID NO: 62, or an active variant or fragment thereof, the RNA-guided nuclease or an active variant or fragment thereof having SEQ ID NO: 20 binds to a target nucleotide sequence adjacent to a PAM sequence as set forth in SEQ ID NO: 146 or 147. It is well known in the art that the PAM sequence specificity for a given nuclease is affected by enzyme concentration (see, e.g., Karvelis et al., (2015) Genome Biol 16: 253), which can be modified by altering the promoter used to express the RGN or the amount of ribonucleoprotein complex delivered to the cell or embryo.

[0219] After recognizing its corresponding PAM sequence, the RGN can cut one or both strands of the target DNA sequence at a specific cleavage site. As used herein, the cleavage site consists of two specific nucleotides within the target DNA sequence, and the strand of the target DNA locus is cut by the RGN between these two nucleotides. The cleavage site can include the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 7th and 8th, or 8th and 9th nucleotides of the PAM in the 5' or 3' direction. In some embodiments, the cleavage site can be more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides away from the PAM in the 5' or 3' direction. Since the RGN can cut the target DNA sequence to cause staggered ends, in some embodiments, the cleavage site is defined according to the distance between the two nucleotides and the PAM on the target strand of the target DNA sequence, and for the non-target strand, it is defined according to the distance between the two nucleotides and the PAM complement.

[0220] IV. Nucleotides encoding RNA-guided nucleases, CRISPR RNAs and / or tracrRNAs

[0221] The present disclosure provides polynucleotides comprising CRISPR RNA, tracrRNA and / or gRNA of the present disclosure and polynucleotides comprising nucleotide sequences encoding RNA-guided nucleases, CRISPR RNA, tracrRNA and / or gRNA of the present disclosure. The polynucleotides of the present disclosure include those comprising or encoding crRNAs comprising CRISPR repeat sequences or active variants or fragments thereof, wherein the CRISPR repeat sequences have any one of the nucleotide sequences of SEQ ID NO: 21-41, or SEQ ID NO: nucleotides 1-17 of 1041 or 1042, or SEQ ID NO: nucleotides 1-22 of 1044 or 1045, which when contained in guide RNAs can direct the sequence-specific binding of the associated RNA-guided nuclease to the target sequence of interest. Also disclosed is a polynucleotide comprising or encoding a tracrRNA having any one of the nucleotide sequences shown in SEQ ID NO: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NO: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045, or an active variant or fragment thereof, which, when included in a guide RNA, can direct the sequence-specific binding of an associated RNA-guided nuclease to a target sequence of interest. Also provided is a polynucleotide encoding an RNA-guided nuclease having any one of the amino acid sequences shown in SEQ ID NO: 1-20, and an active fragment or variant thereof that retains the ability to bind to a target sequence in an RNA-guided sequence-specific manner.

[0222] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising DNA. One of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. These include peptide nucleic acids (PNA), PNA-DNA chimeras, locked nucleic acids (LNA), and phosphorothioate linked sequences. The polynucleotides disclosed herein also include all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, DNA-RNA hybrids, triplex structures, stem-loop structures, and the like.

[0223] In some embodiments, the polynucleotide encoding the RGN of the present disclosure is an mRNA (messenger RNA) molecule. mRNA refers to any polynucleotide that encodes a polypeptide of interest and can be translated in vitro, in vivo, in situ or ex vivo to produce the encoded polypeptide of interest. In embodiments, the essential components of an mRNA molecule include at least a coding region, a 5′UTR, a 3′UTR, a 5′ cap, and a poly-A tail. In embodiments, the mRNA encoding the RGN useful in the methods and compositions of the present disclosure may include one or more structural and / or chemical modifications or changes that confer useful properties on the polynucleotide. For example, one useful property of an mRNA includes a lack of substantial induction of an innate immune response to a cell into which the mRNA is introduced. A "structural" feature or modification refers to a feature or modification in which two or more linked nucleotides are inserted, deleted, duplicated, reversed, or randomized in an mRNA without significant chemical modification of the nucleotides themselves. Because chemical bonds must be broken and rearranged to achieve structural modifications, structural modifications are chemical in nature and are therefore chemical modifications. However, structural modifications will result in different nucleotide sequences. Chemical modification of mRNA may involve the addition of 5-methylcytosine, N1-methylpseudouracil, pseudouracil, 2-thiouridine, 4-thiouridine, 5-methoxyuracil, 2'fluoroguanosine, 2'fluorouridine, 5-bromouridine, 5-(2-carbon methoxyvinyl)uridine, 5-[3(1-E-propyleneamino)]uridine, α-thiocytidine, N6-methyladenosine, 5-methylcytidine, N4-acetylcytidine, 5-formylcytidine or a combination thereof to the mRNA.

[0224] Nucleic acid molecules encoding RGNs can be codon optimized for expression in an organism of interest. A "codon optimized" coding sequence is a polynucleotide coding sequence whose codon usage frequency is designed to mimic the preferred codon usage frequency or transcription conditions of a particular host cell. Due to changes in one or more codons at the nucleic acid level, expression in a particular host cell or organism is enhanced so that the translated amino acid sequence does not change. Nucleic acid molecules can be codon optimized in whole or in part. Codon tables and other references providing preference information for a wide range of organisms are available in the art (for a discussion of codon usage preferred by plants, see, e.g., Campbell and Gowri (1990) Plant Physiol. 92: 1-11). Methods for synthesizing plant-preferred genes or mammalian (e.g., human) codon-optimized coding sequences are available in the art. See, e.g., U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al., (1989) Nucleic Acids Res. 17: 477-498, which are incorporated herein by reference. Non-limiting examples of codon-optimized coding sequences of RGNs of the present disclosure are shown in SEQ ID NOs: 148-167.

[0225] The polynucleotides encoding the RGN, crRNA, tracrRNA and / or gRNA provided herein can be provided in an expression cassette for in vitro expression or expression in a cell, organelle, embryo or organism of interest. The cassette will include 5' and 3' regulatory sequences, which are operably connected to the polynucleotides encoding the RGN, crRNA, tracrRNA and / or gRNA provided herein to allow expression of the polynucleotides. The cassette may additionally include at least one additional gene or genetic element to be co-transformed into an organism. If additional genes or elements are included, each component is operably connected. The term "operably connected" is intended to represent the functional connection between two or more elements. For example, the operably connected between a promoter and a coding region of interest (e.g., the coding region of RGN, crRNA, tracrRNA and / or gRNA) is a functional connection that allows expression of a coding region of interest. The operably connected elements can be continuous or non-continuous. When used to refer to the connection of two protein coding regions, operably connected means that the coding region is located in the same reading frame. Alternatively, additional genes or elements can be provided on multiple expression cassettes. For example, the nucleotide sequence encoding the RGN of the present disclosure can be present on one expression cassette, while the nucleotide sequence encoding the crRNA, tracrRNA or guide RNA can be located on a separate expression cassette. Such an expression cassette has multiple restriction sites and / or recombination sites for inserting a polynucleotide to be under the transcriptional control of the regulatory region. The expression cassette may additionally contain a selectable marker gene.

[0226] The expression cassette will include a transcription (in some embodiments, translation) initiation region (ie, promoter), a polynucleotide encoding RGN-, crRNA-, tracrRNA and / or sgRNA- of the present invention in the 5'-3' direction of transcription, and a transcription (in some embodiments, translation) termination region (ie, termination region) that is functional in an organism of interest. The promoter of the present invention is capable of directing or driving the expression of a coding sequence in a host cell. The regulatory region (eg, promoter, transcriptional regulatory region, and translational termination region) may be endogenous or heterologous to the host cell, or heterologous to each other. As used herein, "heterologous" refers to a sequence derived from an alien species, or, if from the same species, a sequence that is substantially modified in its composition from its native form and / or genomic site by intentional human intervention. As used herein, a chimeric gene comprises a coding sequence that is operably linked to a transcription initiation region heterologous to the coding sequence.

[0227] Convenient termination regions are available from the Ti plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15: 9627-9639.

[0228] Other regulatory signals include, but are not limited to, transcription initiation start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), hereinafter referred to as "Sambrook 11"; Davis et al., eds., (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, NY, and references cited therein.

[0229] In preparing the expression cassette, the various DNA fragments may be manipulated to provide the DNA sequence in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters or linkers may be used to connect the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction, annealing, re-substitution, such as conversion and inversion, may be involved.

[0230] Many promoters can be used in the practice of the present invention. Promoters can be selected according to the desired results. Nucleic acid can be combined with constitutive, inducible, growth phase specific, cell type specific, tissue-preferred, tissue-specific or other promoters to express in the organism of interest. See, e.g., the promoters described in WO 99 / 43838 and U.S. Pat. Nos. 8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; the disclosures of which are incorporated herein by reference.

[0231] For expression in plants, constitutive promoters also include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); and MAS (Velten et al. (1984) EMBO J. 3:2723-2730).

[0232] Examples of inducible promoters are the Adh1 promoter, which can be induced by hypoxia or low temperature stress, the Hsp70 promoter, which can be induced by heat stress, the PPDK promoter, which can be induced by light, and the phosphoenolpyruvate carboxylase promoter. Chemically inducible promoters are also useful, such as the safer inducible In2-2 promoter (U.S. Pat. No. 5,364,780), the auxin-induced Axig1 promoter, the tapetum-specific promoter that is also active in callus (PCT US01 / 22169), the steroid-responsive promoter (see, for example, the estrogen-induced ERE promoter, and Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant Genetics. J. 14(2):247-257), and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0233] Tissue-specific or tissue-preferred promoters can be used for the expression of expression constructs in targeted specific tissues. In certain embodiments, tissue-specific or tissue-preferred promoters are active in plant tissues. Examples of promoters that are developmentally controlled in plants include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, fruits, seeds, or flowers. A "tissue-specific" promoter is a promoter that initiates transcription only in certain tissues. Different from the constitutive expression of a gene, tissue-specific expression is the result of several interaction levels of gene regulation. Therefore, promoters from homologous or closely related plant species can be preferably used to realize efficient and reliable expression of transgenics in specific tissues. In some embodiments, the expression comprises a tissue-preferred promoter. A "tissue-preferred" promoter is a promoter that preferentially initiates transcription, but does not necessarily initiate transcription completely or only in certain tissues.

[0234] In some embodiments, the nucleic acid molecules encoding RGN, crRNA and / or tracrRNA comprise cell type-specific promoters. A "cell type-specific" promoter is a promoter that primarily drives expression of certain cell types in one or more organs. Some examples of plant cells in which cell type-specific promoters that play a role in plants may have primary activity include BETL cells, vascular cells in roots and leaves, stem cells, and stem cells. The nucleic acid molecule may also include a cell type-preferred promoter. A "cell type-preferred" promoter is a promoter that primarily drives most expression, but does not necessarily drive expression completely or only in certain cell types in one or more organs. Some examples of plant cells in which cell type-preferred promoters that play a role in plants may be preferentially active include BETL cells, vascular cells in roots and leaves, stem cells, and stem cells.

[0235] The nucleic acid sequence encoding RGN, crRNA, tracrRNA and / or gRNA can be operably linked to a promoter sequence recognized by a bacteriophage RNA polymerase, for example, for in vitro mRNA synthesis. In such an embodiment, the RNA transcribed in vitro can be purified for use in the methods described herein. For example, the promoter sequence can be a T7, T3 or SP6 promoter sequence, or a variant of a T7, T3 or SP6 promoter sequence. In such an embodiment, the expressed protein and / or RNA can be purified for use in the genome modification methods described herein.

[0236] In certain embodiments, the polynucleotides encoding RGN, crRNA, tracrRNA and / or gRNA may also be linked to a polyadenylation signal (e.g., SV40 polyA signal and other signals that function in plants) and / or at least one transcription termination sequence. In addition, the sequence encoding the RGN may also be linked to a sequence encoding at least one nuclear localization signal, at least one cell penetration domain and / or at least one signal peptide capable of transporting the protein to a specific subcellular location, as described elsewhere herein.

[0237] The polynucleotides encoding RGN, crRNA, tracrRNA and / or gRNA may be present in one or more vectors. "Vector" refers to a polynucleotide composition for transferring, delivering or introducing nucleic acid into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial / mini-chromosomes, transposons and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vectors). The vector may include other expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences), selectable marker sequences (e.g., antibiotic resistance genes), replication origins, etc. For more information, see "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0238] The vector may also include a selectable marker gene for selecting transformed cells. The selectable marker gene is used to select transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds such as glufosinate, bromoxynil, imidazolinone and 2,4-dichlorophenoxyacetate (2,4-D).

[0239] In some embodiments, the expression cassette or vector comprising a sequence encoding an RGN polypeptide may further comprise a sequence encoding crRNA and / or tracrRNA, or crRNA and tracrRNA may be combined to produce gRNA. The sequence encoding crRNA and / or tracrRNA may be operably linked to at least one transcription control sequence for expressing crRNA and / or tracrRNA in an organism or host cell of interest. For example, a polynucleotide encoding crRNA and / or tracrRNA may be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Examples of suitable Pol III promoters include, but are not limited to, mammalian U6, U3, H1 and 7SL RNA promoters and rice U6 and U3 promoters, such as the human U6 promoter shown as SEQ ID NO: 173, and promoters disclosed in U.S. Provisional Application No. 63 / 209,660 filed on June 11, 2021 and PCT International Application No. PCT / US2022 / 032940 filed on June 10, 2022, each of which is incorporated herein by reference in its entirety, including those shown herein as SEQ ID NOs: 553-562.

[0240] As indicated, expression constructs comprising nucleotide sequences encoding RGN, crRNA, tracrRNA and / or gRNA can be used to transform an organism of interest. The transformation method includes introducing a nucleotide construct into an organism of interest. "Introduction" is intended to introduce a nucleotide construct into a host cell so that the construct can enter the interior of the host cell. The method of the present invention does not require a specific method for introducing a nucleotide construct into a host organism, only that the nucleotide construct enters the interior of at least one cell of the host organism. The host cell can be a eukaryotic cell or a prokaryotic cell. In specific embodiments, the eukaryotic host cell is a plant cell, a mammalian cell, an avian cell, or an insect cell. In some embodiments, the eukaryotic cell comprising or expressing the RGN of the present disclosure or modified by the RGN of the present disclosure is a human cell. In some embodiments, the eukaryotic cells comprising or expressing the RGNs of the present disclosure or modified by the RGNs of the present disclosure are cells of hematopoietic origin, such as immune cells (i.e., cells of the innate or adaptive immune system), including but not limited to B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. In some embodiments, the eukaryotic cells comprising or expressing the RGNs of the present disclosure or modified by the RGNs of the present disclosure are eye cells, muscle cells (e.g., skeletal muscle cells), epithelial cells (e.g., lung epithelial cells), diseased cells (e.g., tumor cells).

[0241] Methods for introducing nucleotide constructs into plants and other host cells are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0242] These methods produce transformed organisms, such as plants, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and their progeny. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

[0243] A "transgenic organism" or "transformed organism" or "stably transformed" organism or cell or tissue refers to an organism into which a polynucleotide encoding the RGN, crRNA and / or tracrRNA of the present invention has been incorporated or integrated. It is well known that other exogenous or endogenous nucleic acid sequences or DNA fragments can also be incorporated into the host cell. Agrobacterium and gene gun-mediated transformation remain the two main methods for plant cell transformation. However, transformation of host cells can be carried out by infection, transfection, microinjection, electroporation, microprojection, gene gun or particle bombardment, electroporation, silica / carbon fiber, ultrasound-mediated, PEG-mediated, calcium phosphate coprecipitation, polycationic DMSO technology, DEAE dextran procedure, and viral mediation, liposome mediation, etc. Virus-mediated introduction of polynucleotides encoding RGN, crRNA and / or tracrRNA includes retrovirus-, lentivirus-, adenovirus- and adeno-associated virus-mediated introduction and expression, as well as the use of cauliflower mosaic virus, geminivirus and RNA plant virus.

[0244] Transformation protocols and protocols for introducing polypeptide or polynucleotide sequences into plants may vary depending on the type of host cell targeted for transformation (e.g., a monocot cell or a dicot cell). Transformation methods are known in the art and include those shown in U.S. Pat. Nos.: 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is incorporated herein by reference. See also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12; Bates, GW (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; ​​Christou, P. (1995) Euphytica 85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041-1047; Jones et al. (2005) Plant Methods 1:5.

[0245] Transformation can result in stable or transient incorporation of nucleic acids into cells. "Stable transformation" refers to a nucleotide construct introduced into a host cell that is integrated into the host cell's genome and can be inherited by its progeny. "Transient transformation" refers to a polynucleotide that is introduced into a host cell but not integrated into the host cell's genome.

[0246] Chloroplast transformation methods are known in the art. For example, see Svab et al. (1990) Proc. Nail. Acad. Sci. USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targets the DNA to the plastid genome by homologous recombination. In addition, plastid transformation can be accomplished by transactivating silent plastid-borne transgenes through tissue-preferred expression of nuclear-encoded and plastid-guided RNA polymerases. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7301-7305.

[0247] The cells that have been transformed can be grown into transgenic organisms, such as plants, in a conventional manner. For example, see McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be planted, and with the same transformed plants or different plants pollinated, the resulting hybrids have the constitutive expression of the desired phenotypic characteristics identified. Two or more generations can be planted to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and then seeds are harvested to ensure that the expression of the desired phenotypic characteristics is achieved. In this way, the invention provides transformed seeds (also referred to as "transgenic seeds"), in which the nucleotide construct of the present invention (for example, expression cassette of the present invention) is stably incorporated into its genome.

[0248] Alternatively, transformed cells may be introduced into an organism.These cells may be derived from an organism in which the cells were transformed ex vivo.

[0249] Sequences provided herein can be used for the transformation of any plant species, including but not limited to monocots and dicots. Examples of plants of interest include but are not limited to corn (maize), sorghum, wheat, sunflower, tomato, crucifers, pepper, potato, cotton, rice, soybean, beet, sugarcane, tobacco, barley and rape, Brassica, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamental plants and conifers.

[0250] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Cucumis such as cucumbers, cantaloupes, and cantaloupes. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. In a specific embodiment, the plant of the present invention is a crop (e.g., corn, sorghum, wheat, sunflower, tomato, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugar cane, tobacco, barley, rapeseed, etc.).

[0251] As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures, plant callus, plant masses, and complete plant cells in a plant or a part of a plant, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, spikes, cobs, shells, stems, roots, root tips, anthers, etc. that can regenerate plants. Cereals refer to mature seeds produced by commercial growers for purposes other than planting or breeding the species. Offspring, variants, and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts comprise the polynucleotides introduced. Further provided are processed plant products or byproducts retaining sequences disclosed herein, for example, including soybean meal.

[0252] Polynucleotides encoding or comprising RGN, crRNA and / or tracrRNA can also be used to transform any prokaryotic species, including, but not limited to, Archaea and bacteria (e.g., Bacillus species, Klebsiella species, Streptomyces species, Rhizobium species, Escherichia species, Pseudomonas species, Salmonella species, Shigella species, Vibrio species, Yersinia species, Mycoplasma species, Agrobacterium, Lactobacillus species).

[0253] Polynucleotides encoding or comprising RGN, crRNA and / or tracrRNA can be used to transform any eukaryotic species, including but not limited to animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoeba, algae, and yeast.

[0254] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian, insect or avian cells or target tissues. Such methods can be used to administer nucleic acids encoding RGN system components to cells in culture or host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to cells. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1: 13-26 (1994).

[0255] Non-viral delivery methods for nucleic acids include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virus particles, and drug-enhanced DNA uptake. For example, lipofection is described in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355), and lipofection reagents are commercially available (e.g., Transfectam TM and Lipofectin TM). Cationic and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Can be delivered to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). The preparation of lipid:nucleic acid complexes (including targeted liposomes, such as immunolipid complexes) is well known to those skilled in the art (e.g., see Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0256] Nucleic acid is delivered using a system based on RNA or DNA virus, using a highly evolved process to target specific cells in the virus body and transport the viral payload to the nucleus. Viral vectors can be directly administered to patients (in vivo), or they can be used for in vitro treatment of cells, and the modified cells can be optionally administered to patients (ex vivo). Traditional virus-based systems can include retroviruses, slow viruses, adenoviruses, adeno-associated viruses and herpes simplex virus vectors for gene transfer. Retroviruses, slow viruses and adeno-associated viruses gene transfer methods can be integrated into the host genome, usually resulting in long-term expression of the transgenic inserted. In addition, high transduction efficiency is observed in many different cell types and target tissues.

[0257] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, thereby expanding the potential target cell population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells, typically producing high viral titers. Therefore, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packaging capacity of up to 6-10 kb of foreign sequences. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the therapeutic gene into the target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66:1635-1640 (1992); Sommnerfelt et al., J. Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., J. Viral. 65:2220-2224 (1991); PCT / US94 / 05700).

[0258] In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are able to achieve very high transduction efficiencies in many cell types and do not require cell division. Using such vectors, high titers and expression levels have been achieved. Such vectors can be mass-produced in relatively simple systems. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, e.g., for in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). The construction of recombinant AAV vectors is described in many publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., 1. Viral. 63:03822-3828 (1989). Packaging cells are generally used to form viral particles that can infect host cells. Such cells include 293 cells for packaging adenovirus, and ψJ2 cells or PA317 cells for packaging retrovirus.

[0259] Viral vectors used in gene therapy are typically produced by generating cell lines that package nucleic acid vectors into viral particles. Vectors typically contain the minimal viral sequences required for packaging and subsequent integration into the host, with other viral sequences replaced by an expression cassette that expresses the polynucleotide. The missing viral functions are typically provided in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome that are required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid that encodes the other AAV genes (i.e., rep and cap) but lacks the ITR sequences.

[0260] The cell line can also be infected with adenovirus as a helper virus. The helper virus promotes the replication of the AAV vector and the expression of AAV genes from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large quantities. Adenovirus contamination can be reduced by, for example, heat treatment, because adenovirus is more sensitive to heat treatment than AAV. Other methods of delivering nucleic acids to cells are well known to those skilled in the art. For example, see US20030087817, which is incorporated herein by reference.

[0261] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, the cell is transfected because it naturally exists in the subject. In some embodiments, the transfected cell is taken from the subject. In some embodiments, the cell is derived from a cell taken from the subject, such as a cell line. In some embodiments, the cell line can be a mammalian, insect or avian cell. A variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CVI, RPTE, A10, T24, 182, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4.COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB / 3T3 mouse embryonic fibroblasts, 3T3Swiss, 3T3-Ll, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293,BxPC3,C3H-10Tl / 2,C6 / 36,Cal-27,CHO,CHO-7,CHO-IR,CHO-Kl,CHO-K2,CHO-T,CHODhfr- / -,COR-L23,COR-L23 / CPR,COR-L235010,CORL23 / R23,COS-7,COV-434,CML Tl,CMT,CT26,D17,DH82,DU145,DuCaP,EL4,EM2,EM3,EMT6 / AR1,EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, lY cells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48,MC-38,MCF-7,MCF-l0A,MDA-MB-231,MDA-MB-468,MDA-MB-435,MDCKII,MDCKII,MOR / 0.2R,MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines can be obtained from various sources known to those skilled in the art (see, e.g., American Type Culture Collection (ATCC) (Manassas, Va.)). .

[0262] In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines comprising one or more vector-derived sequences. In some embodiments, cells transiently transfected with components of the RGN system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA) and modified by the activity of the RGN system are used to establish new cell lines comprising cells containing the modifications but lacking any other exogenous sequences. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells, are used to evaluate one or more test compounds.

[0263] In some embodiments, one or more vectors described herein are used to produce non-human transgenic animals or transgenic plants. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, hamster, rabbit, cow, or pig. In some embodiments, the transgenic animal is a bird, such as a chicken or duck. In some embodiments, the transgenic animal is an insect, such as a mosquito or a tick.

[0264] V. Variants and fragments of polypeptides and polynucleotides

[0265] The present disclosure provides active variants and fragments of naturally occurring (i.e., wild-type) RNA-guided nucleases whose amino acid sequences are set forth in any one of SEQ ID NOs: 1-20, as well as active variants and fragments of naturally occurring CRISPR repeat sequences, such as any one of the sequences set forth in SEQ ID NOs: 21-41, or nucleotides 1-17 set forth in SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 set forth in SEQ ID NOs: 1044 or 1045, and active variants and fragments of naturally occurring tracrRNA, such as any one of the sequences set forth in SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NOs: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045, and polynucleotides encoding the same.

[0266] Although the activity of a variant or fragment may vary compared to a polynucleotide or polypeptide of interest, the variants and fragments should retain the functionality of the polynucleotide or polypeptide of interest. For example, a variant or fragment may have increased activity, decreased activity, a different activity profile, or any other altered activity compared to a polynucleotide or polypeptide of interest.

[0267] Fragments and variants of naturally occurring RGN polypeptides, such as those disclosed herein, will retain sequence-specific RNA-guided DNA binding activity. In certain embodiments, fragments and variants of naturally occurring RGN polypeptides, such as those disclosed herein, will retain nuclease activity (single-stranded or double-stranded).

[0268] Fragments and variants of naturally occurring CRISPR repeat sequences, such as those disclosed herein, when part of a guide RNA (including tracrRNA), will retain the ability to bind and direct an RNA-guided nuclease (complexed with the guide RNA) to a target sequence (e.g., a target DNA sequence) in a sequence-specific manner.

[0269] Fragments and variants of naturally occurring tracrRNA, such as those disclosed herein, when part of a guide RNA (including a CRISPR RNA), will retain the ability to direct an RNA-guided nuclease (complexed with the guide RNA) to a target sequence (e.g., a target DNA sequence) in a sequence-specific manner.

[0270] The term "fragment" refers to a portion of a polynucleotide or polypeptide sequence of the invention. "Fragments" or "biologically active portions" include polynucleotides that contain a sufficient number of contiguous nucleotides to retain biological activity (i.e., when included in a guide RNA, bind to and direct the RGN to a target nucleotide sequence in a sequence-specific manner). "Fragments" or "biologically active portions" include polypeptides that contain a sufficient number of contiguous amino acid residues to retain biological activity (i.e., when complexed with a guide RNA, bind to a target sequence in a sequence-specific manner). Fragments of RGN proteins include fragments that are shorter than the full-length sequence due to the use of alternative downstream start sites. A biologically active portion of an RGN protein can be, for example, a polypeptide comprising 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more consecutive amino acid residues of any one of SEQ ID NOs: 1 to 20. Such biologically active portions can be prepared by recombinant techniques and assessed for sequence-specific RNA-guided DNA binding activity. A biologically active fragment of a CRISPR repeat sequence can comprise at least 8 contiguous amino acids of any one of SEQ ID NOs: 21-41, or nucleotides 1-14 of SEQ ID NO: 1040, nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, nucleotides 1-19 of SEQ ID NO: 1043, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045. A biologically active portion of a CRISPR repeat sequence can be, for example, a polynucleotide comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of any one of SEQ ID NOs: 21-41, or nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045.A biologically active portion of a tracrRNA can be, for example, a polynucleotide comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more consecutive nucleotides of any one of SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NOs: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045.

[0271] In general, "variant" means a substantially similar sequence. For polynucleotides, variants include deletions and / or additions of one or more nucleotides at one or more internal sites within a natural polynucleotide and / or replacements of one or more nucleotides at one or more sites in a natural polynucleotide. As used herein, "natural" or "wild-type" polynucleotides or polypeptides include naturally occurring nucleotide sequences or amino acid sequences, respectively. For polynucleotides, conservative variants include those sequences that encode the native amino acid sequence of a gene of interest due to the degeneracy of the genetic code. These naturally occurring allelic variants can be identified using well-known molecular biology techniques, such as using polymerase chain reaction (PCR) and hybridization techniques, as described below. Variant polynucleotides also include synthetically derived polynucleotides, such as those produced using site-directed mutagenesis but still encoding a polypeptide or polynucleotide of interest. Generally, variants of a particular polynucleotide disclosed herein have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by the sequence alignment programs and parameters described elsewhere herein.

[0272] The variant of a specific polynucleotide disclosed herein (i.e., a reference polynucleotide) can also be assessed by comparing the sequence identity percentage between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The sequence alignment program and parameters described elsewhere herein can be used to calculate the sequence identity percentage between any two polypeptides. When assessing any given polynucleotide pair disclosed herein by comparing the sequence identity percentage shared by two polypeptides encoded by them, the sequence identity percentage between two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.

[0273] In certain embodiments, the polynucleotides of the present disclosure encode RNA-guided nuclease polypeptides comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the amino acid sequences shown in SEQ ID NOs: 1-20.

[0274] Biologically active variants of the RGN polypeptides of the invention may differ by as few as about 1-15 amino acid residues, as few as about 1-10 amino acid residues, such as about 6-10 amino acid residues, as few as about 5 amino acid residues, as few as about 4 amino acid residues, as few as about 3 amino acid residues, as few as about 2 amino acid residues, or as few as about 1 amino acid residue. In specific embodiments, the polypeptide may comprise an N-terminal or C-terminal truncation which may comprise a deletion of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more amino acids from the N or C terminus of the polypeptide.

[0275] In certain embodiments, a polynucleotide of the present disclosure comprises or encodes a CRISPR repeat sequence comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the nucleotide sequences set forth as SEQ ID NOs:21-41, or nucleotides 1-17 of SEQ ID NOs:1041 or 1042, or nucleotides 1-22 of SEQ ID NOs:1044 or 1045.

[0276] The polynucleotides of the present disclosure may comprise or encode a tracrRNA comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NO: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045.

[0277] Biologically active variants of the CRISPR repeat sequences or tracrRNA of the invention may differ by as few as about 1-15 nucleotides, as few as about 1-10 nucleotides, such as about 6-10 nucleotides, as few as 5 nucleotides, as few as 4 nucleotides, as few as 3 nucleotides, as few as 2 nucleotides, or as few as 1 nucleotide. In specific embodiments, the polynucleotide may comprise a 5' or 3' truncation, which may comprise a deletion of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110 or more nucleotides from the 5' or 3' end of the polynucleotide.

[0278] It is recognized that the RGN polypeptides, CRISPR repeat sequences and tracrRNA provided herein can be modified to generate variant proteins and polynucleotides. Artificially designed changes can be introduced by applying site-directed mutagenesis techniques. Alternatively, natural, unknown or unidentified polynucleotides and / or polypeptides that are structurally and / or functionally related to the sequences disclosed herein that fall within the scope of the present invention can also be identified. Conservative amino acid substitutions can be made in non-conserved regions that do not alter the function of the RGN protein. Alternatively, modifications can be made to increase the activity of the RGN.

[0279] Variant polynucleotides and proteins also encompass sequences and proteins derived from mutagenesis and recombination procedures such as DNA shuffling. Through such procedures, one or more of the different RGN proteins disclosed herein (e.g., SEQ ID NOs: 1-20) are manipulated to create new RGN proteins with desired properties. In this way, a library of recombinant polynucleotides is generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding domains of interest can be shuffled between the RGN sequences provided herein and other known RGN genes to obtain proteins encoding domains of interest with improved properties of interest (such as increased K in the case of enzymes). m) of a protein. Strategies for such DNA shuffling are known in the art. See, e.g., Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri et al. (1998) Nature 391: 288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458. "Shuffled" nucleic acids are nucleic acids produced by a shuffling procedure (such as any shuffling procedure described herein). Shuffled nucleic acids are produced by recombining (physical or virtual) two or more nucleic acids (or strings), for example in an artificial and optionally recursive manner. Typically, one or more screening steps are used in the shuffling process to identify the nucleic acids of interest; the screening step can be performed before or after any recombination step. In some (but not all) shuffling embodiments, it is desirable to perform multiple rounds of recombination before selection to increase the diversity of the pool to be screened. The entire process of recombination and selection is optionally repeated recursively. Depending on the context, shuffling can refer to the entire process of recombination and selection, or alternatively, can simply refer to the recombination portion of the entire process.

[0280] As used herein, in the context of two polynucleotides or polypeptide sequences, "sequence identity" or "identity" refers to the same residues in two sequences when aligned on a specified comparison window to obtain maximum correspondence. When using the percentage of sequence identity to refer to a protein, it should be recognized that the non-identical residue positions are usually different due to conservative amino acid substitutions, wherein the amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and therefore the functional properties of the molecule will not be changed. When the sequences are different in conservative substitutions, the percentage of sequence identity can be adjusted upward to correct the conservative nature of the substitution. Sequences that differ due to such conservative substitutions are referred to as having "sequence similarity" or "similarity". The method for making such adjustments is well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Therefore, for example, when the same amino acid is assigned 1 point and non-conservative substitutions are assigned 0 points, conservative substitutions are assigned a score between 0 and 1. The scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0281] As used herein, "sequence identity percentage" refers to a value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) to achieve optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the sequence identity percentage.

[0282] Unless otherwise indicated, the sequence identity / similarity values ​​provided herein refer to the values ​​obtained using GAP version 10 using the following parameters: using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmp scoring matrix to calculate the % identity and % similarity of nucleotide sequences; using GAP Weight of 8 and Length Weight of 2 and the BLOSUM62 scoring matrix to calculate the % identity and % similarity of amino acid sequences; or any equivalent programs thereof. "Equivalent program" refers to any sequence comparison program that, for any two query sequences, generates an alignment with the same number of nucleotide or amino acid residue matches and the same percentage sequence identity when compared to the corresponding alignment generated by GAP version 10.

[0283] When two sequences are aligned using a defined amino acid substitution matrix (e.g., BLOSUM62), a gap existence penalty, and a gap extension penalty, the two sequences are "optimally aligned" when the similarity score is scored so as to obtain the highest possible score for the sequence pair. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well known in the art, such as in Dayhoff et al. (1978) "A model of evolutionary change in proteins.", "Atlas of Protein Sequence and Structure," Vol.5, Suppl.3 (ed. MO Dayhoff), pp.345-352. Natl. Biomed. Res. Found., Washington, DC and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919. The BLOSUM62 matrix is ​​often used as a default scoring substitution matrix in sequence alignment procedures. Gap existence penalty is imposed for introducing a single amino acid gap in one of the alignment sequences, and gap extension penalty is imposed for each additional empty amino acid position inserted into the opened gap. Alignment is defined by the amino acid position of each sequence that the alignment begins and ends, and is optionally defined by inserting one or more gaps in one or both sequences to obtain a score as high as possible. Although optimal alignment and scoring can be completed manually, the process is promoted by using a computer-implemented alignment algorithm, for example, gap BLAST 2.0, is recorded in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, and is provided to the public on the website of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Optimal alignments (including multiple alignments) can be prepared using, for example, PSI-BLAST, available through www.ncbi.nlm.nih.gov and described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

[0284] For an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds to" the position of that residue in the reference sequence that is paired in the alignment. "Position" is represented by a number that identifies each amino acid in the reference sequence in turn according to its position relative to the N-terminus in the reference sequence. Because deletions, insertions, truncations, fusions, etc. must be considered when determining the optimal alignment, in general, the number of amino acid residues in a test sequence determined by counting only from the N-terminus is not necessarily the same as the number of their corresponding positions in the reference sequence. For example, in the case of a deletion in the aligned test sequence, there will be no amino acid corresponding to the position in the reference sequence at the site of the deletion. If there is an insertion in the aligned reference sequence, the insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions, there may be stretches of amino acids in the reference sequence or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0285] VI. Antibodies

[0286] Also included are antibodies against the RGN polypeptides of the invention or ribonucleoproteins comprising the RGN polypeptides, including polypeptides having any one of the amino acid sequences shown in SEQ ID NOs: 1-20, or active variants or fragments thereof. Methods for producing antibodies are well known in the art (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and U.S. Pat. No. 4,196,265). These antibodies can be used in kits for detecting and isolating RGN polypeptides or ribonucleoproteins. Thus, the present disclosure provides kits comprising antibodies that specifically bind to the polypeptides or ribonucleoproteins described herein, including, for example, polypeptides having any one of the amino acid sequences shown in SEQ ID NOs: 1-20.

[0287] VII. RGN systems and ribonucleoprotein complexes for binding target sequences of interest and methods for preparing the same

[0288] The present disclosure provides a system for binding to a target sequence of interest (e.g., a target DNA sequence), wherein the system includes at least one RNA-guided nuclease or a nucleotide sequence encoding the nuclease and one or more guide RNAs capable of forming a complex (ribonucleoprotein complex) with an RGN polypeptide. The guide RNA hybridizes with the non-target strand of the target sequence of interest and also forms a complex with the RGN polypeptide, thereby directing the RGN polypeptide to bind to the target DNA sequence. In some of these embodiments, the RGN comprises any one of the amino acid sequences shown in SEQ ID NOs: 1-20, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence having any one of the nucleotide sequences shown in SEQ ID NOs: 21-41, or nucleotides 1-14 of SEQ ID NOs: 1040, nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, nucleotides 1-19 of SEQ ID NOs: 1043, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045, or an active variant or fragment thereof. In certain embodiments, the guide RNA comprises a tracrRNA having any one of the nucleotide sequences shown in SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NO: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045, or an active variant or fragment thereof. The guide RNA of the system can be a single guide RNA or a dual guide RNA. In certain embodiments, the system includes an RNA-guided nuclease heterologous to the guide RNA, wherein the RGN and the guide RNA are not found complexed with each other (i.e., bound to each other) in nature.

[0289] The system provided herein for binding to a target sequence of interest can be a ribonucleoprotein complex, which is at least one RNA molecule combined with at least one protein. The ribonucleoprotein complex provided herein includes at least one guide RNA as an RNA component and an RNA-guided nuclease as a protein component. Such ribonucleoprotein complexes can be purified from cells or organisms that naturally express RGN polypeptides and have been transformed to express specific guide RNAs specific to target sequences of interest. Alternatively, the ribonucleoprotein complex can be purified from cells or organisms that have been transformed with polynucleotides encoding RGN polypeptides and guide RNAs (or polynucleotides comprising guide RNAs) and cultured under conditions that allow expression of RGN polypeptides and guide RNAs. Therefore, a method for making RGN polypeptides or RGN ribonucleoprotein complexes is provided. Such methods include culturing cells comprising a nucleotide sequence encoding an RGN polypeptide under conditions that express an RGN polypeptide (in some embodiments, a guide RNA), and in some embodiments, culturing cells comprising a nucleotide sequence encoding or comprising a guide RNA. The RGN polypeptide or RGN ribonucleoprotein can then be purified from the lysate of the cultured cells. In some embodiments, the nucleotide sequence encoding the RGN polypeptide comprises mRNA (messenger RNA). In some embodiments, a method of assembling an RNP complex comprises combining one or more guide RNAs of the disclosure and one or more RGN polypeptides of the disclosure under conditions suitable for the formation of the RNP complex.

[0290] Methods for purifying RGN polypeptides or RGN ribonucleoprotein complexes from biological sample lysates are known in the art (e.g., size exclusion and / or affinity chromatography, 2D-PAGE, HPLC, reverse phase chromatography, immunoprecipitation). In specific methods, the RGN polypeptide is recombinantly produced and comprises a purification tag to aid in its purification, including, but not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly (NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG (e.g., 3X FLAG tag), HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 10xHis, biotin carboxyl carrier protein (BCCP), and calmodulin. Typically, immobilized metal affinity chromatography is used to purify the tagged RGN polypeptide or RGN ribonucleoprotein complex. It will be appreciated that other similar methods known in the art, including other forms of chromatography or, for example, immunoprecipitation, may be used, alone or in combination.

[0291] An "isolated" or "purified" polypeptide or its biologically active portion is substantially or substantially free of components that normally accompany or interact with the polypeptide in its naturally occurring environment. Thus, an isolated or purified polypeptide is substantially free of other cellular materials or culture media when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Proteins that are substantially free of cellular materials include protein preparations with less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating proteins. When the protein of the present invention or its biologically active portion is recombinantly produced, the optimal culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest. Similarly, an "isolated" polynucleotide or nucleic acid molecule is removed from its naturally occurring environment. An isolated polynucleotide is substantially free of chemical precursors or other chemicals when chemically synthesized or removed from a genomic locus by cleavage of a phosphodiester bond. An isolated polynucleotide can be part of a vector, a composition of matter, or can be contained within a cell, as long as the cell is not the original environment of the polynucleotide.

[0292] Specific methods provided herein for binding and / or cleaving target nucleic acid molecules comprising a target sequence of interest involve the use of RGN ribonucleoprotein complexes assembled in vitro. The in vitro assembly of RGN ribonucleoprotein complexes can be performed using any method known in the art, wherein the RGN polypeptide is contacted with the guide RNA under conditions that allow the RGN polypeptide to bind to the guide RNA. As used herein, "contacting", "contacted" refers to bringing together the components of the desired reaction under conditions suitable for carrying out the desired reaction. The RGN polypeptide can be purified from a biological sample, cell lysate, or culture medium, produced by in vitro translation, or produced by chemical synthesis. The guide RNA can be purified from a biological sample, cell lysate, or culture medium, transcribed in vitro, or chemically synthesized. The RGN polypeptide and the guide RNA can be contacted in a solution (e.g., a buffered saline solution) to allow for the in vitro assembly of the RGN ribonucleoprotein complex.

[0293] VIII. Methods for Binding, Cleavage or Modification of Target Nucleic Acid Molecules

[0294] The present disclosure provides methods for binding, cutting and / or modifying a target nucleic acid molecule of interest (e.g., target DNA) comprising a target sequence. The method comprises delivering a system comprising at least one guide RNA or a polynucleotide encoding the guide RNA and at least one RGN polypeptide or a polynucleotide encoding the RGN polypeptide to a target sequence or a cell, an organelle, or an embryo comprising the target sequence. In some of these embodiments, the RGN comprises any one of the amino acid sequences shown in SEQ ID NO: 1-20, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising any one of the nucleotide sequences shown in SEQ ID NO: 21-41, or nucleotides 1-17 of SEQ ID NO: 1041 or 1042, or nucleotides 1-22 of SEQ ID NO: 1044 or 1045, or an active variant or fragment thereof. In a specific embodiment, the guide RNA comprises a tracrRNA comprising any one of the nucleotide sequences shown in SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NO: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045, or an active variant or fragment thereof. The guide RNA of the system can be a single guide RNA or a dual guide RNA.

[0295] The RGN of the system can be a nuclease-inactivated RGN, with nickase activity, or can be a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a base editing polypeptide, such as a cytosine deaminase or an adenine deaminase. In other embodiments, the RGN fusion protein comprises a reverse transcriptase. In other embodiments, the RGN fusion protein comprises a polypeptide that recruits a functional nucleic acid repair complex member, such as a member of a nucleotide excision repair (NER) or transcription-coupled nucleotide excision repair (TC-NER) pathway (Wei et al., 2015, PNAS USA 112 (27): E3495-504; Troelstra et al., 1992, Cell 71: 939-953; Marnef et al., 2017, J Mol Biol 429 (9): 1277-1288), as described in U.S. Provisional Application No. 63 / 332,486 filed on April 19, 2022, which is incorporated by reference in its entirety. In some embodiments, the RGN fusion protein comprises CSB (van den Boom et al., 2004, J Cell Biol 166(1):27-36; van Gool et al., 1997, EMBO J16(19):5955-65; an example thereof is shown in SEQ ID NO:563), which is a member of the TC-NER (nucleotide excision repair) pathway and plays a role in the recruitment of other members. In further embodiments, the RGN fusion protein comprises an active domain of CSB, such as the acidic domain of CSB, which comprises amino acid residues 356-394 of SEQ ID NO:563 (Teng et al., 2018, Nat Commun 9(1):4115).

[0296] In certain embodiments, the RGN and / or guide RNA is heterologous to the cell, organelle, or embryo into which the RGN and / or guide RNA (or a polynucleotide encoding at least one of the RGN and guide RNA) is introduced.

[0297] In those embodiments in which the method includes delivering a polynucleotide encoding a guide RNA and / or an RGN polypeptide, cells or embryos can then be cultured under conditions expressing the guide RNA and / or the RGN polypeptide. In various embodiments, the method includes contacting the target nucleic acid molecule with an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex may include an RGN that is nuclease-inactivated or has nickase activity. In some embodiments, the RGN of the ribonucleoprotein complex is a fusion polypeptide comprising a base editing polypeptide. In certain embodiments, the method includes introducing the RGN ribonucleoprotein complex into a cell, an organelle, or an embryo comprising a target nucleic acid molecule. The RGN ribonucleoprotein complex may be purified from a biological sample, recombinantly produced and subsequently purified, or assembled in vitro as described herein. In those embodiments in which the RGN ribonucleoprotein complex in contact with a target nucleic acid molecule or an organelle or an embryo has been assembled in vitro, the method may also include assembling the complex in vitro before contacting the target nucleic acid molecule, cell, organelle, or embryo.

[0298] Purified or in vitro assembled RGN ribonucleoprotein complexes can be introduced into cells, organelles, or embryos using any method known in the art, including but not limited to electroporation. Alternatively, RGN polypeptides and / or polynucleotides encoding or comprising guide RNAs can be introduced into cells, organelles, or embryos using any method known in the art, such as electroporation.

[0299] After delivery to or contact with a target nucleic acid molecule or a cell, organelle, or embryo comprising the target nucleic acid molecule, the guide RNA directs the RGN to bind to a target sequence within the target nucleic acid molecule in a sequence-specific manner. In those embodiments in which the RGN has nuclease activity, the RGN polypeptide cleaves the target sequence of interest after binding. The target DNA sequence can then be modified by endogenous repair mechanisms such as non-homologous end joining or homology-directed repair with a provided donor polynucleotide.

[0300] Methods for measuring the binding of RGN polypeptides to target sequences are known in the art, including chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter gene assays, microplate capture and detection assays. Similarly, methods for measuring the cleavage or modification of target nucleic acid molecules comprising target sequences are also known in the art, including in vitro or in vivo cleavage assays, wherein cleavage is confirmed using PCR, sequencing or gel electrophoresis, with or without appropriate labels (e.g., radioisotopes, fluorescent substances) attached to the target sequence to facilitate detection of degradation products. Alternatively, the nicking triggered exponential amplification reaction (NTEXPAR) assay can be used (see, e.g., Zhang et al. (2016) Chem. Sci. 7: 4951-4957). In vivo cleavage can be assessed using the Surveyor assay (Guschin et al. (2010) Methods Mol Biol 649: 247-256).

[0301] In some embodiments, the method involves the use of a single type of RGN complexed with more than one guide RNA. The more than one guide RNA can target different regions of a single gene or can target multiple genes.

[0302] In embodiments where a donor polynucleotide is not provided, double-strand breaks introduced by the RGN polypeptide can be repaired by a non-homologous end joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, repair of double-strand breaks can result in modification of the target sequence. As used herein, "modification" with respect to a nucleic acid molecule refers to a change in the nucleotide sequence of a nucleic acid molecule, which can be a deletion, insertion or substitution of one or more nucleotides, or a combination thereof. Modification of a target nucleic acid molecule comprising a target sequence can result in expression of an altered protein product or inactivation of a coding sequence.

[0303] In those embodiments where there is a donor polynucleotide, the donor sequence in the donor polynucleotide can be integrated into the target nucleotide sequence or exchanged with the target nucleotide sequence during the process of repairing the introduced double-strand break, thereby resulting in the introduction of an exogenous donor sequence. Therefore, the donor polynucleotide comprises a donor sequence that is desired to be introduced into the target sequence of interest. In some embodiments, the donor sequence changes the original target nucleotide sequence so that the newly integrated donor sequence will not be recognized and cut by the RGN. The integration of the donor sequence can be enhanced by including a flanking sequence (referred to herein as a "homology arm") in the donor polynucleotide, and the homology arm has substantial sequence identity with the sequence flanking the target nucleotide sequence, thereby allowing a homology-directed repair process. In some embodiments, the length of the homology arm is at least 50 base pairs, at least 100 base pairs, and at most 2000 base pairs or more, and has at least 90%, at least 95% or more sequence homology with the corresponding sequence in the target nucleotide sequence.

[0304] In those embodiments in which the RGN polypeptide introduces double-stranded staggered breaks, the donor polynucleotide can comprise a donor sequence flanked by compatible overhangs, thereby allowing for direct ligation of the donor sequence to the cleaved target nucleotide sequence comprising the overhangs by non-homologous repair processes during double-strand break repair.

[0305] In those embodiments where the method involves the use of an RGN as a nickase (i.e., capable of cleaving only a single strand of a double-stranded polynucleotide), the method can include introducing two RGN nickases that target the same or overlapping target sequences and cleave different strands of the polynucleotide. For example, an RGN nickase that cleaves only the plus (+) strand of a double-stranded polynucleotide and a second RGN nickase that cleaves only the minus (-) strand of a double-stranded polynucleotide can be introduced.

[0306] In various embodiments, a method of binding to a target nucleotide sequence and detecting the target sequence is provided, wherein the method comprises introducing into a cell, organelle, or embryo at least one guide RNA or polynucleotide encoding the guide RNA and at least one RGN polypeptide or polynucleotide encoding the RGN polypeptide, expressing the guide RNA and / or RGN polypeptide (if the coding sequence is introduced), wherein the RGN polypeptide is a nuclease-inactive RGN and further comprises a detectable label, and the method further comprises detecting the detectable label. The detectable label can be fused to the RGN as a fusion protein (e.g., a fluorescent protein), or can be a small molecule that binds to or is bound to the RGN polypeptide, which can be detected visually or otherwise.

[0307] Also provided herein are methods for regulating the expression of a target gene of interest comprising a target sequence or a gene regulated by a target sequence. The method comprises introducing at least one guide RNA or a polynucleotide encoding the guide RNA and at least one RGN polypeptide or a polynucleotide encoding the RGN polypeptide into a cell, an organelle, or an embryo, expressing the guide RNA and / or the RGN polypeptide (if the coding sequence is introduced), wherein the RGN polypeptide is a nuclease-inactivated RGN. In some of these embodiments, the nuclease-inactivated RGN is a fusion protein comprising an expression regulator domain (i.e., an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain) as described herein.

[0308] The disclosure also provides a method for binding and / or modifying a target nucleic acid molecule of interest comprising a target sequence. The method includes delivering a fusion polypeptide (e.g., cytosine deaminase or adenine deaminase) comprising at least one guide RNA or a polynucleotide encoding the guide RNA and at least one RGN of the present invention and a base editing polypeptide, or a polynucleotide encoding the fusion polypeptide to a target sequence or a cell, organelle, or embryo comprising the target sequence.

[0309] In some embodiments where a fusion polypeptide comprising an RGN and a base editing polypeptide is utilized, binding of the fusion protein to the target sequence results in modification of nucleotides adjacent to the target sequence. The nucleobases adjacent to the target sequence that are modified by the deaminase can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5' or 3' end of the target sequence.

[0310] Those of ordinary skill in the art will appreciate that any of the methods disclosed herein can be used to target a single target sequence or multiple target sequences. Thus, these methods include using a single RGN polypeptide in combination with multiple different guide RNAs, which can target multiple different sequences within a single gene and / or multiple genes. Also contemplated herein are methods that introduce multiple different guide RNAs in combination with multiple different RGN polypeptides. These guide RNAs and guide RNA / RGN polypeptide systems can target multiple different sequences within a single gene and / or multiple genes.

[0311] In one aspect, the present invention provides a kit comprising any one or more elements disclosed in the above methods and compositions, including crRNA, tracrRNA, guide RNA, RGN and / or polynucleotides encoding them, cells and RGN systems. In some embodiments, the kit includes a vector system and instructions for using the kit. In some embodiments, the vector system includes (a) a first regulatory element, which is operably connected to a DNA sequence encoding a guide RNA and one or more insertion sites for inserting the guide sequence into the upstream of the encoded guide RNA, wherein when expressed, the guide RNA directs the sequence-specific binding of the RGN complex to the target sequence in the eukaryotic cell, wherein the RGN complex includes an RGN enzyme compounded with a guide RNA polynucleotide; and / or (b) a second regulatory element, which is operably connected to an enzyme coding sequence encoding the RGN enzyme, and the enzyme coding sequence includes a nuclear localization sequence. In some embodiments, the kit also includes a homologous recombination template polynucleotide. These elements can be provided alone or in combination, and can be provided in any suitable container, such as a vial, a bottle or a test tube.

[0312] In some embodiments, the kit includes instructions in one or more languages. In some embodiments, the kit includes one or more reagents, and the reagent is used in the method utilizing one or more elements described herein. Reagent can be provided in any suitable container. For example, the kit can provide one or more reaction or storage buffers. Reagent can be provided in a form that can be used for a specific assay, or in a form that needs to add one or more other components before use (such as concentrate or lyophilized form). Buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer and combinations thereof. In some embodiments, buffer is alkaline. In some embodiments, the pH of buffer is about 7 to about 10.

[0313] In one aspect, the present invention provides methods of using one or more elements of an RGN system. The RGN system of the present invention provides an effective method for modifying a target polynucleotide. The RGN system of the present invention has a variety of uses, including modifying (e.g., deleting, inserting, shifting, inactivating, activating, base editing) a target polynucleotide in a variety of cell types. Therefore, the RGN system of the present invention has a wide range of applications, for example, gene therapy, drug screening, disease diagnosis and prognosis. An exemplary RGN system or RGN complex includes an RGN enzyme complexed with a guide sequence capable of binding to a target sequence.

[0314] IX. Target Polynucleotides

[0315] In one aspect, the invention provides methods for modifying a target polynucleotide comprising a target sequence or modifying the expression of a target polynucleotide in a eukaryotic cell, the method being in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling cells or cell groups from humans or non-human animals or plants (including microalgae) and modifying the cells. Cultivation can be performed at any stage in vitro. Cells can even be reintroduced into non-human animals or plants (including microalgae).

[0316] Using natural variability, plant breeders combine the most useful genes to obtain desired qualities, such as yield, quality, consistency, cold tolerance and insect resistance. These desired qualities also include growth, day length preference, temperature requirements, start date of flowering or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, tolerance to various environmental factors, including drought, heat, humidity, cold, wind and unfavorable soil conditions including high salinity. The sources of these useful genes include local or exotic varieties, heirloom varieties, wild plant relatives and induced mutations, such as treating plant materials with mutagens. Using the present invention, a new tool for inducing mutations is provided for plant breeders. Therefore, those skilled in the art can analyze genomes to find the source of useful genes, and use the present invention to induce the production of useful genes in varieties with desired characteristics or traits, more accurately than previous mutagens, thereby accelerating and improving plant breeding programs.

[0317] The target polynucleotide of the RGN system can be any polynucleotide endogenous or exogenous to a eukaryotic cell. For example, the target polynucleotide can be a polynucleotide located in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or non-coding DNA). Without being bound by theory, the target strand of the target sequence should be adjacent to the PAM (protospacer adjacent motif); that is, a short sequence recognized by the RGN system. The exact sequence and length requirements of the PAM vary depending on the RGN used, but the PAM is usually a 2-7 base pair sequence adjacent to the protospacer sequence (i.e., the target sequence).

[0318] The target polynucleotides of the RGN system can include many disease-related genes and polynucleotides and signal transduction biochemical pathway-related genes and polynucleotides. Examples of target polynucleotides include sequences related to signal transduction biochemical pathways, for example, signal transduction biochemical pathway-related genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. "Disease-related" genes or polynucleotides refer to any genes or polynucleotides that produce transcription or translation products at abnormal levels or in abnormal forms in cells from tissues affected by the disease compared to tissues or cells of non-disease controls. It can be a gene with an abnormally high expression level; it can be a gene with an abnormally low expression level, wherein the altered expression is associated with the occurrence and / or progression of the disease. Disease-related genes also refer to genes with mutations or genetic variations that directly cause the cause of the disease or are in a state of linkage disequilibrium (e.g., causal mutations) with genes that cause the cause of the disease. The products of transcription or translation may be known or unknown, and may also be at normal or abnormal levels. In some embodiments, the disease may be an animal disease. In some embodiments, the disease may be a bird disease. In other embodiments, the disease may be a mammalian disease. In further embodiments, the disease may be a human disease. Examples of human disease-associated genes and polynucleotides are available from the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information at the National Library of Medicine (Bethesda, Md.), available on the World Wide Web.

[0319] Although the RGN system is particularly useful because it is relatively easy to target genomic sequences of interest, there remains a question of what RGNs can do to address causal mutations. One approach is to produce a fusion protein between an RGN (e.g., an inactive or nickase variant of an RGN) and an active domain of a base editing enzyme or a base editing enzyme (such as a cytosine deaminase or adenine deaminase base editor) (U.S. Patent No. 9,840,699, which is incorporated herein by reference). In some embodiments, the method includes contacting a DNA molecule comprising a target sequence with (a) a fusion protein comprising an RGN of the invention or its nickase variant and a base editing polypeptide (such as a deaminase); and (b) contacting the fusion protein of (a) to a gRNA targeting the target sequence; wherein the DNA molecule is contacted with the fusion protein and gRNA in an effective amount and under conditions suitable for nucleobase deamination. In some embodiments, the target DNA sequence includes a sequence associated with a disease or disorder, and wherein nucleobase deamination produces a sequence unrelated to the disease or disorder. In some embodiments, the target DNA sequence is located in an allele of a crop plant, wherein a particular allele of a trait of interest results in a plant with lower agronomic value. Nucleobase deamination produces an allele that improves the trait and increases the agronomic value of the plant.

[0320] In some embodiments, the target DNA sequence includes a T→C or A→G point mutation associated with a disease or condition, and wherein deamination of the mutated C or G base produces a sequence not associated with the disease or condition. In some embodiments, deamination corrects a point mutation in a sequence associated with a disease or condition.

[0321] In some embodiments, the sequence associated with the disease or condition encodes a protein, wherein deamination introduces a stop codon into the sequence associated with the disease or condition, resulting in truncation of the encoded protein. In some embodiments, contacting is performed in a subject susceptible to, suffering from, or diagnosed with a disease or condition. In some embodiments, the disease or condition is a disease associated with a point mutation or a single base mutation in the genome. In some embodiments, the disease is a genetic disease, cancer, metabolic disease, or lysosomal storage disease.

[0322] X. Pharmaceutical Compositions and Methods of Treatment

[0323] The present invention provides pharmaceutical compositions comprising the RGN polypeptides disclosed herein, active variants and fragments thereof, and polynucleotides encoding the same, the crRNAs disclosed herein, active variants and fragments thereof, or polynucleotides encoding the same, the tracrRNAs disclosed herein, active variants and fragments thereof, or polynucleotides encoding the same, the gRNAs disclosed herein, or polynucleotides encoding the gRNAs, the systems disclosed herein, or cells comprising any of the RGN polypeptides or polynucleotides encoding the RGNs, the gRNAs or polynucleotides encoding the gRNAs, or the RGN systems, and pharmaceutically acceptable carriers.

[0324] A pharmaceutical composition is a composition for preventing, alleviating in intensity, curing or otherwise treating a target condition or disease, comprising an active ingredient (i.e., an RGN polypeptide, a polynucleotide encoding an RGN, a gRNA, a polynucleotide encoding a gRNA, an RGN system, or a cell comprising any of them) and a pharmaceutically acceptable carrier.

[0325] As used herein, a "pharmaceutically acceptable carrier" refers to a material that does not cause significant irritation to an organism and does not abrogate the activity and properties of an active ingredient (i.e., an RGN polypeptide, a polynucleotide encoding an RGN, a gRNA, a polynucleotide encoding a gRNA, an RGN system, or a cell comprising any of them). The carrier must be of sufficiently high purity and sufficiently low toxicity to make it suitable for administration to a subject being treated. The carrier may be inert, or may have a pharmaceutical benefit. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid fillers, diluents, or encapsulating materials that are suitable for administration to humans or other vertebrates. In some embodiments, a pharmaceutically acceptable carrier is not naturally occurring. In some embodiments, a pharmaceutically acceptable carrier and an active ingredient do not coexist in nature.

[0326] The pharmaceutical composition used in the method of the present disclosure can be formulated with suitable carriers, excipients and other agents providing suitable transfer, delivery, tolerance, etc. A variety of suitable preparations are known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st edition, 2005). Suitable preparations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles (such as LIPOFECTIN vesicles) containing lipids (cations or anions), lipid nanoparticles, DNA conjugates, anhydrous absorption pastes, water-in-oil and oil-in-water emulsions, emulsion polyethylene glycol carbowax (polyethylene glycol of various molecular weights), semisolid gels and semisolid mixtures containing polyethylene glycol carbowax. Pharmaceutical compositions for oral or parenteral use can be prepared into dosage forms suitable for unit doses of a single dose of matching active ingredients. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0327] The present disclosure provides a pharmaceutical composition comprising a lipid-based formulation comprising an active ingredient (i.e., a guide RNA and / or RGN, or a polynucleotide comprising or encoding them). In some embodiments, the lipid-based formulation comprises a liposome. In some embodiments, the lipid-based formulation comprises a lipid nanoparticle (LNP). In some embodiments, the active ingredient is encapsulated in a lipid particle and / or is located on the surface of a lipid particle. In some embodiments, the active ingredient is covalently linked to the lipid particle. In some embodiments, the active ingredient is non-covalently associated with the lipid particle. Covalent bonding includes electron sharing in a chemical bond. Non-covalent interactions include dispersed electromagnetic interactions, such as hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds.

[0328] In some embodiments, the active ingredient is encapsulated in lipid particles. The term "encapsulation" refers to encapsulation, surrounding or encapsulation. With respect to the preparation of the compounds of the present disclosure, encapsulation can be substantial, complete or partial. The term "substantially encapsulated" or "substantially encapsulated" refers to pharmaceutical compositions of the present disclosure or active ingredients greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or more can be enclosed, surrounded or encapsulated in a delivery agent (e.g., liposome or LNP). The term "partial encapsulation" or "partial encapsulation" refers to pharmaceutical compositions of the present disclosure or active ingredients less than 50%, 40%, 30%, 20%, 10% or less can be enclosed, surrounded or encapsulated in a delivery agent. Encapsulation can be determined by using fluorescence and / or electron microscopy to measure the escape or activity of pharmaceutical compositions of the present disclosure or active ingredients. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or more of a pharmaceutical composition or active ingredient of the present disclosure is encapsulated in a delivery agent (e.g., liposomes or LNPs).

[0329] Liposomes are spherical vesicle structures consisting of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have attracted widespread attention as drug delivery vehicles because they are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their cargo across biological membranes and the blood-brain barrier (BBB) ​​(see, e.g., Spuch and Navarro (2011) Journal of drug delivery 2011).

[0330] Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Although liposomes form spontaneously when a lipid film is mixed with an aqueous solution, formation can also be accelerated by applying force in the form of shaking using a homogenizer, ultrasonic generator, or extrusion device (see, e.g., Spuch and Navarro (2011) Journal of drug delivery 2011).

[0331] Conventional liposome preparations are mainly composed of natural phospholipids and phospholipids, such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine and monosialoganglioside. In some embodiments, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases the stability of liposomes.

[0332] Additives can be added to liposomes to change their structure and properties. In some embodiments, cholesterol and / or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of the cargo within the liposome. In some embodiments, cholesterol is added to conventional liposome preparations to reduce the rapid release of encapsulated active ingredients (i.e., guide RNA and / or RGN, or polynucleotides comprising or encoding them) into plasma. In some embodiments, liposomes are prepared by hydrogenated lecithin or lecithin, cholesterol and cetyl phosphate. In some embodiments, the average liposome vesicle size is adjusted to about 50 or 100nm.

[0333] In some embodiments, Trojan horse liposomes (also known as molecular Trojan horses or pegylated immunoliposomes) can be used in pharmaceutical compositions to deliver active ingredients across the BBB (described on the World Wide Web at cshprotocols.cshlp.org / content / 2010 / 4 / pdb.prot5407.long). Without being bound by any theory, it is believed that neutral lipid particles with specific antibodies conjugated to their surfaces allow for crossing the BBB by endocytosis. In some embodiments, pharmaceutical compositions comprising Trojan horse liposomes can be used to deliver active ingredients (i.e., guide RNAs and / or RGNs, or polynucleotides comprising or encoding them) to the brain by intravascular injection.

[0334] In some embodiments, the liposomes include stable nucleic acid-lipid particles (SNALP) (see, e.g., Morrissey et al. (2005) Nature Biotechnology 23(8): 1002-1007; Zimmerman et al. (2006) Nature 441: 111-114). SNALPs include a mixture of cationic lipids and fusogenic lipids and are coated with polyethylene glycol (PEG), allowing cells to take up the active ingredient cargo and release it in the endosomal. In some embodiments, SNALPs are a class of LNPs that include ionizable lipids that are cationic at low pH (e.g., DLinDMA), neutral helper lipids, cholesterol, and diffusible polyethylene glycol (PEG) lipids. In some embodiments, the SNALP formulation includes the following lipids: 3-N-(-methoxypoly(ethylene glycol) 2000)carbamoyl-1,2-dimyristyloxy-propylamine (PEG-cDMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. In some embodiments, SNALP includes synthetic cholesterol, dipalmitoylphosphatidylcholine (DOPC), PEG-cDMA, and DLinDMA (see, e.g., Geisbert et al. (2010) Lancet 375: 1896-1905). In some embodiments, SNALP includes synthetic cholesterol, DSPC, PEG-cDMA, and DLinDMA (see, e.g., Judge et al. (2009) J. Clin. Invest. 119: 661-673). In some embodiments, the size of the SNALP liposomes is about 80-100 nm. SNALP has been used as an effective delivery molecule for highly vascularized HepG2-derived liver tumors (see, e.g., Li et al. (2012) Gene Therapy 19:775-780).

[0335] Not bound by any one theory, in the formulation of SNALP, ionizable lipids are used for condensation of lipids with active ingredients (e.g., nucleic acid molecules) during particle formation. When positively charged under increasingly acidic endosomal conditions, ionizable lipids can mediate the fusion of SNALP with endosomal membranes, thereby enabling the release of active ingredients into the cytoplasm. PEG-lipids can stabilize particles and reduce aggregation during formulation, and can subsequently provide a neutral hydrophilic exterior, thereby improving pharmacokinetic properties. In some embodiments, SNALP liposomes are formulated by DLinDMA and PEG-cDMA with DSPC, cholesterol and active ingredients, using 25:1 lipid:active ingredient ratio and 48:40:10:2 molar ratio of cholesterol:DLinDMA:DSPC:PEG-cDMA.

[0336] In some embodiments, the pharmaceutical composition of the present disclosure includes LNP. In some embodiments, lipids can be formulated with the active ingredients of the present disclosure to form LNP. LNP includes multiple lipid molecules, which are physically associated with each other by intermolecular forces. In some embodiments, LNP includes liposomes. In some embodiments, LNP is different from liposomes in that it does not have a continuous lipid bilayer. In some embodiments, LNP includes solid particles with a mixture of solid and liquid lipids. In some embodiments, LNP includes dendrimer lipid nanoparticles (DLNP), SNALP and lipid-like nanoparticles (LLNP). In general, "nanoparticles" refer to any particles with a diameter less than 1000 nanometers (nm). In some embodiments, the diameter of the nanoparticles is 500nm or less. In some embodiments, the diameter of the nanoparticles ranges from 25nm to 200nm, or 100nm or less. In some embodiments, the diameter of the nanoparticles ranges from 35nm to 60nm. In some embodiments, LNP includes lipid particles with a size of about 1 to about 100nm.

[0337] LNP includes four kinds of components: ionizable cationic lipid, fusion zwitterionic phospholipid, cholesterol and pegylated (PEG) lipid.In some embodiments, ionizable cationic lipid component is compounded with negatively charged polynucleotide and strengthens endosome escape.In some embodiments, phospholipid component plays the effect of modifying lipid bilayer structure.In some embodiments, cholesterol component helps to stabilize LNP.In some embodiments, PEG lipid component reduces LNP aggregation and non-specific uptake.

[0338] Ionizable cationic lipids that can be used in LNPs include: 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP); DLinDMA; 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinK-DMA); 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA); 5A2-SC8 (Zhou et al. (2016) Proc. Natl Acad. Sci. USA 113:520–525); C12-200 (Love et al. (2010) Proc. Natl Acad. Sci. USA 107:1864–1869); 246C10 (Kim et al. (2021) Sci Adv 7(9):eabf4398); cKK-E12 (Fenton et al. (2016) Advanced Materials 28(15):2939-2943); 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA); and dilinoleyl methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA; Jayaraman et al. (2012) Angew Chem Int Ed Engl. 51(34):8529-8533). Cationic lipids are further described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, and WO2008103276, U.S. Pat. Nos. 7,893,302 and 7,404,969, and U.S. Patent Publication No. US20100036115, each of which is herein incorporated by reference in its entirety.

[0339] Zwitterionic phospholipids that can be used in LNPs include DSPC, DOPE, and DOPC.

[0340] PEG lipids that can be used in LNPs include: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG); (3-o-[2"-(methoxypolyethylene glycol 2000) succinyl] -1,2-dimyristoyl-sn-ethylene glycol (PEG-S-DMG); R-3-[(ω-methoxy-poly(ethylene glycol) 2000) carbamoyl] -1,2-dimyristoyloxypropyl-3-amine (PEG-C-DOMG); and C16PEG-ceramide. In some embodiments, LNPs include DLinKC2-DMA or C12-200: DSPC: cholesterol: PEG-DMG in a molar ratio of 50:10:38.5:1.5 (see, e.g., Basha et al. (2011) Molecular Therapy 19(12):2186-2200). In some embodiments, the LNP comprises 26.5:20:52:1.5 ionizable lipid:DOPE:cholesterol:PEG lipid (see, e.g., Han et al. (2022) Sci Adv 8(3):eabj6901; Kim et al. (2021) Sci Adv 7(9):eabf4398). PEG lipids are further described in WO2012099755. In some embodiments, the proportion of PEG in the LNP formulation can be increased or decreased and / or the carbon chain length of the PEG lipid can be modified from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the LNP formulation.

[0341] In some embodiments, the charge of LNP is considered.Cationic lipids can be combined with negatively charged lipids to induce a non-double-layer structure that is conducive to intracellular delivery. Because charged LNP can be rapidly removed from circulation after intravenous injection, an ionizable cationic lipid with a pKa value lower than 7 is developed (see, e.g., Basha et al. (2011) Molecular Therapy 19 (12): 2186-2200). Negatively charged polymers (such as, polynucleotides) can be loaded into LNP at low pH values ​​(e.g., pH 4), where ionizable lipids show positive charge. However, at physiological pH values, LNP shows a low surface charge compatible with a longer circulation time.

[0342] Preparation of LNPs and encapsulation of active ingredients are described, for example, in Basha et al. (2011) Molecular Therapy 19(12):1286-2200; Han et al. (2022) Sci Adv 8(3):eabj6901; Kim et al. (2021) Sci Adv 7(9):eabf4398; Finn et al. (2018) Cell Reports 22:2227–2235; Wei et al. (2020) Nature Communications 11:3232; WO2011127255; and WO2008103276. Lipids are commercially available (e.g., from Tekmira Pharmaceuticals, Vancouver, Canada; Avanti Polar Lipids, Inc., Alabaster, AL) or can be synthesized (e.g., Kim et al. (2021) Sci Adv 7(9):eabf4398). The synthesis of cationic lipids is also described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, and WO201021865. Cholesterol is commercially available (e.g., from Sigma-Aldrich, St Louis, MO).

[0343] In some embodiments, encapsulation can be performed by dissolving a lipid mixture comprising a cationic lipid (e.g., Dlin-DMA): a phospholipid (e.g., DSPC, DOPE): cholesterol: a PEG-lipid (e.g., a molar ratio of 40:10:40:10) in ethanol. The active ingredient (e.g., a polynucleotide comprising or encoding a guide RNA or RGN of the present disclosure) can be dissolved in an acidic buffer (e.g., citrate, acetate), pH 3 or 4. In some embodiments, the lipid solution and active ingredient solution can be mixed using a microfluidic system (Chen et al. (2012) J. Amer. Chem. Soc. 134: 6948-6951; e.g., NanoAssemblr from Precision Nanosystems) or by adding the lipid solution dropwise to the active ingredient solution. Removal of ethanol and neutralization of the formulation buffer can be performed by dialysis against phosphate buffered saline (PBS) using a dialysis cassette (e.g., 3500 molecular weight cutoff cassette from Life Technologies), e.g., for 16 hours or overnight. Dynamic light scattering can be used to assess LNP size, polydispersity index (PDI), and zeta potential. The size of the LNPs can be determined by a quantitation system such as Quant-it TM The encapsulation efficiency of active ingredients (such as RNA) is determined by the assay of RibogreenAssay (Thermo Fisher). In some embodiments where the encapsulated active ingredient is a polynucleotide, the polynucleotide can be extracted from the eluted nanoparticles and quantified at 260 nm. The LNP pKa can be assessed using the 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS) assay (Zhang et al. (2011) Langmuir 27(5):1907–1914). In some embodiments, the final lipid: active ingredient weight ratio includes 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, and 5:1.

[0344] In some embodiments where the pharmaceutical composition comprises a ribonucleoprotein (RNP) complex (i.e., RGN and guide RNA) encapsulated in LNP, an ethanol solution of lipid is mixed with an RNP solution at physiological pH (e.g., PBS buffer; Wei et al. (2020) Nature Communications 11: 3232), and an additional permanent cationic lipid (e.g., 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) is added to allow the formation of LNPs comprising RNPs. In some embodiments, the permanent cationic lipid accounts for 10 to 20 mol% of the total lipids in the LNP.

[0345] In some embodiments, the LNP formulations described herein may further comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US2005 / 0222064.

[0346] In some embodiments, LNP compositions are biodegradable because they do not accumulate to cytotoxic levels in vivo at therapeutically effective doses. LNP formulations can be improved by replacing cationic lipids with biodegradable cationic lipids, known as rapidly eliminated lipid nanoparticles (reLNPs). In some embodiments, the rapid metabolism of rapidly eliminated lipids can increase the tolerability and therapeutic index of LNP by an order of magnitude, from a 1 mg / kg dose in rats to a 10 mg / kg dose. The addition of enzymatically degradable ester bonds can improve the degradation and metabolic profiles of the cationic component while still maintaining the activity of the reLNP formulation. The ester bond can be located inside the lipid chain, or can ultimately be located at the end of the lipid chain. An internal ester bond can replace any carbon in the lipid chain.

[0347] In some embodiments, the LNP compositions do not induce an innate immune response that would result in significant adverse reactions at therapeutic dose levels. In some embodiments, the LNP compositions provided herein do not induce toxicity at therapeutic dose levels.

[0348] In some embodiments, the active ingredient (i.e., guide RNA and / or RGN, or polynucleotides comprising or encoding them) is formulated into solid lipid nanoparticles. Solid lipid nanoparticles (SLN) can be spherical with an average diameter between 10 and 1000 nm. SLN has a solid lipid core matrix that can dissolve lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. In further embodiments, the lipid nanoparticles can be self-assembling lipid polymer nanoparticles (see, e.g., Zhang et al. (2008) ACS Nano 2 (8): 1696-1702).

[0349] In some embodiments, lipid-based formulations including active ingredients (i.e., guide RNAs and / or RGNs, or polynucleotides comprising or encoding them) can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a specific release pattern to achieve a therapeutic outcome.

[0350] In some embodiments, the lipid-based formulation comprising the active ingredient (i.e., guide RNA and / or RGN, or polynucleotides comprising or encoding them) comprises at least one controlled release coating. The controlled release coating comprises: (Colorcon Inc., Harleysville, PA); polyvinylpyrrolidone / vinyl acetate copolymer; polyvinylpyrrolidone; hydroxypropyl methylcellulose; hydroxypropyl cellulose; hydroxyethyl cellulose; EUDRAGIT (Evonik, Essen, Germany); EUDRAGIT (Evonik, Essen, Germany); and cellulose derivatives, such as aqueous ethylcellulose dispersions ( and Colorcon Inc., Harleysville, PA). In some embodiments, the controlled release and / or targeted delivery formulation may include at least one degradable polyester, which may contain polycationic side chains. Degradable polyesters include poly(serine esters), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline esters), and combinations thereof. In some embodiments, the degradable polyester may include a PEG conjugate to form a PEGylated polymer.

[0351] In some embodiments, LNP formulations can be prepared so that they passively or actively target different cell types in the body, including hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. (2010) Mol Ther. 18:1357-1364; Song et al. (2005) Nat Biotechnol. 23:709-717; Judge et al. (2009) J Clin Invest. 119:661-673; Kaufmann et al. (2010) Microvasc Res 80:286-293; Santel et al. (2006) Gene Ther 13:1222-1234; Santel et al. (2006) Gene Ther 13:1360-1370; Gutbier et al. (2010) Pulm Pharmacol. Ther. 23:334-344; Basha et al. (2011) Mol. Ther. 19:2186-2200; Fenske and Cullis (2008) Expert Opin Drug Deliv. 5:25-44; Peer et al. (2008) Science 319:627-630; Peer and Lieberman (2011) Gene Ther. 18:1127-1133; all of which are incorporated herein by reference in their entirety). An example of passive targeting of agents to hepatocytes includes DLin-DMA, DLin-KC2-DMA, and MC3-based lipid nanoparticle formulations, which have been shown to bind to apolipoprotein E and promote binding and uptake of these agents into hepatocytes in vivo (Akinc et al. (2010) Mol Ther. 18:1357-1364).

[0352] LNP formulations can also be selectively targeted by expressing different ligands on their surface, such as folic acid, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeting methods (Kolhatkar et al. (2011) Curr Drug Discov Technol. 8:197-206; Musacchio and Torchilin (2011) Front Biosci. 16:1388-1412; Yu et al. (2010) Mol Membr Biol. 27:286-298; Patil et al. (2008) Crit Rev Ther Drug Carrier Syst. 25:1-61; Benoit et al. (2011) Biomacromolecules. 12:2708-2714; Zhao et al. (2008) Expert Opin Drug Deliv. 5:309-319; Akinc et al. (2010) Mol Membr Biol. 27:286-298; Patil et al. (2008) Crit Rev Ther Drug Carrier Syst. 25:1-61; Benoit et al. (2011) Biomacromolecules. 12:2708-2714; Zhao et al. (2008) Expert Opin Drug Deliv. 5:309-319; Akinc et al. (2010) Mol Ther. 18: 1357-1364; Srinivasan et al. (2012) Methods Mol Biol. 820: 105-116; Ben-Arie et al. (2012) Methods Mol Biol. 757: 497-507; Peer, D (2010) J of controlled release 148 (1): 63-68; Peer et al. (2007) Proc Natl Acad Sci USA. 104: 4095-4100; Kim et al. (2011) Methods Mol Biol. 721: 339-353; Subramanya et al. (2010) Mol Ther. 18: 2028-2037; Song et al. (2005) Nat Biotechnol. 23: 709-717; Peer et al. (2008) Science 319:627-630; Peer and Lieberman (2011) Gene Ther. 18:1127-1133; each of which is incorporated herein by reference in its entirety).

[0353] In some embodiments, the active ingredient (i.e., guide RNA and / or RGN, or polynucleotides comprising or encoding them) can be encapsulated into LNPs, which can then be encapsulated into polymers, polymer matrices, hydrogels, and / or surgical sealants described herein and / or known in the art. In some embodiments, the polymer, hydrogel, or surgical sealant comprises: poly(lactic-co-glycolic acid (PLGA); ethylene vinyl acetate (EVAc); poloxamer; (Nanotherapeutics, Inc. Alachua, FL); (Halozyme Therapeutics, San Diego CA); surgical sealants such as fibrinogen polymers (Ethicon Inc., Cornelia, GA) and (Baxter International, Inc Deerfield, IL); PEG-based sealants; and (Baxter International, Inc. Deerfield, IL).

[0354] LNPs and LNP formulations are further described in, e.g., U.S. Patent Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658; European Patent Nos. 1766035; 1519714; 1781593; and 1664316.

[0355] In some embodiments, where cells comprising or modified with the disclosed RGNs, gRNAs, RGN systems, or polynucleotides encoding them are administered to a subject, the cells are administered in the form of a suspension together with a pharmaceutically acceptable carrier. One skilled in the art will recognize that a pharmaceutically acceptable carrier for use in a cell composition will not include a buffer, compound, cryopreservative, preservative, or other agent in an amount that substantially interferes with the viability of the cells to be delivered to the subject. Formulations comprising cells may include, for example, an osmotic buffer that allows for maintenance of cell membrane integrity, and optional nutrients to maintain cell viability or enhance implantation after administration. Such formulations and suspensions are known to those skilled in the art and / or can be adjusted for use with the cells described herein using routine experimentation.

[0356] The cell composition may also be emulsified or in the form of a liposomal composition, as long as the emulsification process does not adversely affect cell viability. The cells and any other active ingredients may be mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient and in an amount suitable for use in the methods of treatment described herein.

[0357] Other agents included in the cell composition can include pharmaceutically acceptable salts of each component. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as, acetic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also come from inorganic bases (such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide) and organic bases (such as, isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0358] Physiologically tolerable and pharmaceutically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any substance other than the active ingredient and water, or contain buffers such as sodium phosphate, saline or both at physiological pH, such as phosphate buffered saline. In addition, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol and other solutes. The liquid composition can also contain a liquid phase other than water and not including water. Examples of such additional liquid phases are glycerol, vegetable oils such as cottonseed oil and water-oil emulsions. The amount of the active compound used in the cell composition to effectively treat a particular disorder or condition can depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0359] The RGN polypeptides, guide RNAs, RGN systems, or polynucleotides encoding them disclosed herein can be formulated with pharmaceutically acceptable excipients (such as carriers, solvents, stabilizers, adjuvants, diluents, etc.), depending on the specific mode of administration and dosage form. In some embodiments, these pharmaceutical compositions are formulated to achieve a physiologically compatible pH, and depending on the formulation and route of administration, the pH ranges from about 3 to about 11, about 3 to about 7. In some embodiments, the pH can be adjusted to a range of about pH 5.0 to about pH 8. In some embodiments, the composition may include a therapeutically effective amount of at least one compound described herein, and one or more pharmaceutically acceptable excipients. In some embodiments, the composition comprises a combination of compounds described herein, or includes a second active ingredient that can be used to treat or prevent bacterial growth (for example, but not limited to, an antibacterial or antimicrobial agent), or includes a combination of agents disclosed herein.

[0360] Suitable excipients include, for example, carrier molecules, including large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactivated viral particles. Other exemplary excipients may include antioxidants (such as, but not limited to, ascorbic acid), chelating agents (such as, but not limited to, EDTA), carbohydrates (such as, but not limited to, dextrins, hydroxyalkyl cellulose, and hydroxyalkyl methyl cellulose), stearic acid, liquids (such as, but not limited to, oils, water, saline, glycerol, and ethanol), wetting agents or emulsifiers, pH buffer substances, and the like.

[0361] In some embodiments, the preparation is provided in a unit dose or multi-dose container, such as a sealed ampoule and vial, and can be stored under freeze-dried (lyophilized) conditions, requiring the addition of a sterile liquid carrier, such as saline, water for injection, semi-liquid foam or gel, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the aforementioned types. In some embodiments, the active ingredient is dissolved in a buffered liquid solution, which is frozen in a unit dose or multi-dose container and then thawed for injection or refrigerated storage / stabilization until use.

[0362] The therapeutic agent can be included in a controlled release system. In order to prolong the effect of the drug, it is usually necessary to slow down the absorption of the drug from subcutaneous, intrathecal or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with poor water solubility. The absorption rate of the drug depends on its dissolution rate, which can depend on crystal size and crystalline form. Alternatively, the delayed absorption of the drug form administered parenterally can be achieved by dissolving or suspending the drug in an oil carrier. In some embodiments, long-term sustained-release implants can be particularly suitable for treating chronic diseases. Long-term sustained-release implants are well known to those of ordinary skill in the art.

[0363] Provided herein are methods for treating a disease in a subject in need thereof. The method comprises administering to a subject in need thereof an effective amount of an RGN polypeptide of the present disclosure or an active variant or fragment thereof or a polynucleotide encoding them, a gRNA of the present disclosure or a polynucleotide encoding the gRNA, an RGN system of the present disclosure, or a cell modified by or comprising any of these compositions.

[0364] In some embodiments, treatment includes in vivo gene editing by administering RGN polypeptides, gRNA or RGN systems of the present disclosure or polynucleotides encoding them. In some embodiments, treatment includes ex vivo gene editing, wherein cells are subjected to ex vivo gene modification with RGN polypeptides, gRNA or RGN systems of the present disclosure or polynucleotides encoding them, and the modified cells are then administered to the subject. In some embodiments, genetically modified cells are derived from subjects to whom modified cells are subsequently administered, and transplanted cells are referred to herein as autologous cells. In some embodiments, genetically modified cells are derived from different subjects (i.e., donors) of the same species as the subjects (i.e., recipients) to whom modified cells are administered, and transplanted cells are referred to herein as allogeneic. In some embodiments described herein, cells can be expanded in culture before being administered to subjects in need.

[0365] In some embodiments, the disease treated with the composition of the present disclosure is a disease that can be treated with immunotherapy, such as with chimeric antigen receptor (CAR) T cells. Such diseases include, but are not limited to, cancer. In some embodiments, the disease treated with the composition of the present disclosure is related to a causal mutation. As used herein, a "causal mutation" refers to a specific nucleotide, nucleotide, or nucleotide sequence in a genome that causes the severity or presence of a disease or condition in a subject. The correction of a causal mutation results in an improvement in at least one symptom caused by a disease or condition. In some embodiments, the causal mutation is adjacent to the PAM site recognized by the RGN disclosed herein. The causal mutation can be corrected with an RGN disclosed herein or a fusion polypeptide comprising an RGN disclosed herein and a base editing polypeptide (i.e., a base editor). Non-limiting examples of diseases associated with causal mutations include cystic fibrosis, Hurler syndrome, Friedreich's Ataxia, Huntington's disease, and sickle cell disease. Table 6 lists other non-limiting examples of genes and mutations associated with diseases, and more examples are available from the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University (Bethesda, Md.) and the National Center for Biotechnology Information at the U.S. National Library of Medicine (Bethesda, Md.), available on the World Wide Web.

[0366] In some embodiments, a method for treating a disease in a subject in need thereof comprises creating an induced pluripotent stem cell (iPSC) or isolating a mesenchymal stem cell from a subject, contacting the iPSC or mesenchymal stem cell with any of the RGN polypeptides, systems, compositions or pharmaceutical compositions disclosed herein, to genetically modify a target nucleic acid molecule within the cell, differentiating the modified iPSC or modified mesenchymal stem cell into a genetically modified mature cell or a precursor thereof, and administering the genetically modified mature cell or a precursor thereof to the subject. In some embodiments, the iPSC or mesenchymal stem cell is an autologous or allogeneic cell. In some embodiments, the iPSC or mesenchymal stem cell is derived from a donor of a human leukocyte antigen (HLA) that is perfectly matched to the subject. In some embodiments, prior to administering the modified cells, the subject is administered myeloablative therapy.

[0367] Patient-specific iPS cells can be created using any method known in the art, including but not limited to, the method described in Takahashi and Yamanaka, Cell 126 (4): 663-76, 2006. For example, the creation step may include: a) isolating somatic cells, such as skin cells or fibroblasts, from a subject; and b) introducing a set of pluripotency-related genes into somatic cells to induce the cells to become pluripotent stem cells. The set of pluripotency-related genes may be one or more genes selected from the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT, and LIN28. Mesenchymal stem cells can be separated according to any method known in the art, such as from the patient's bone marrow or peripheral blood. For example, a bone marrow aspirate can be collected in a syringe containing heparin. Cells can be washed and centrifuged in Percoll. Cells can be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger MF, Mackay AM, Beck SC et al., Science 1999; 284: 143-147).

[0368] The genetically modified cells of the present disclosure administered to a subject include autologous and allogeneic cells. Allogeneic cells refer to cells from a donor (i.e., the individual from whom the genetically modified cells are derived). Autologous cells refer to cells from a subject receiving treatment (i.e., the recipient of the genetically modified cells). Due to the risk of transplant rejection, efforts are made to optimize the degree of major histocompatibility complex (MHC) / human leukocyte antigen (HLA) matching between donor tissue and recipient. HLA is located on the surface of cells and helps the body identify itself and non-self so that the body can attack foreign entities such as bacteria and viruses. HLA typing of donor tissue and recipient involves determining the genotypes of six HLA antigens or alleles between the donor and recipient to assess the degree of six HLA matching. HLA alleles generally refer to two at each of HLA-A, HLA-B and HLA-DR loci, or one at each of HLA-A, HLA-B and HLA-C loci and one at each of HAL-DRB1, HLA-DQB1 and HLA-DPB1 loci (see, e.g., Kawase et al., 2007, Blood 110: 2235-2241). In some embodiments, 4 out of 6 HLA matches between donor and recipient are sufficient to administer cells from the donor to the recipient. In some embodiments, 5 out of 6 HLA matches between donor and recipient are sufficient to administer cells from the donor to the recipient. In some embodiments, 6 out of 6 HLA matches between donor and recipient are sufficient to administer cells from the donor to the recipient. In general, 4 / 6, 5 / 6 or 6 / 6 HLA matches are the standard for clinical care. When all 6 HLA matches between donor and recipient, the match is referred to as a perfect match.

[0369] As used herein, "treatment" or "treating" or "alleviation" or "improvement" are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or preventive benefit. Therapeutic benefit refers to any treatment-related improvement or effect on one or more diseases, conditions or symptoms under treatment. In order to obtain preventive benefit, the composition can be applied to a subject at risk of developing a specific disease, condition or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition or symptom may not yet be manifested. In some embodiments, treatment can be performed after the occurrence of one or more symptoms and / or after the disease is diagnosed. In specific embodiments, treatment can be performed in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of the disease. For example, susceptible individuals can be treated before symptoms appear (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment can also be continued after symptoms subside, for example, to prevent or delay their prevention or recurrence.

[0370] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following: the subject and disease condition being treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., and these factors can be easily determined by a person of ordinary skill in the art. The specific dosage may vary depending on one or more of the following: the specific agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, and the delivery system carrying it.

[0371] The term "administering" refers to placing an active ingredient into a subject by a method or route that causes the introduced active ingredient to be at least partially localized at a desired site (such as a site of injury or repair), thereby producing a desired effect. In some embodiments, the disclosure provides methods comprising delivering RGN polypeptides, nucleic acid molecules, ribonucleoprotein complexes, vectors, pharmaceutical compositions, and / or gRNAs described herein. In some embodiments, the disclosure also provides cells produced by such methods, and organisms (such as animals or plants) comprising or produced by such cells. In some embodiments, the RGN polypeptides and / or nucleic acid molecules described herein are delivered to cells in combination with a guide sequence (and optionally complexed with a guide sequence).

[0372] In those embodiments in which cells are administered, the cells can be administered by any appropriate route so as to be delivered to a desired location in the subject, wherein at least a portion of the implanted cells or cell components remain viable. The survival period of the cells after administration to the subject can be as short as a few hours, such as twenty-four hours, to a few days, to as long as several years, or even the patient's lifetime, i.e., long-term implantation. For example, in some aspects described herein, an effective amount of photoreceptor cells or retinal progenitor cells is administered by a systemic administration route, such as an intraperitoneal or intravenous route.

[0373] In some embodiments, administration includes administration by viral delivery. Viral vectors comprising nucleic acids encoding RGN polypeptides, ribonucleoprotein complexes, or vectors disclosed herein can be directly administered to patients (i.e., in vivo), or they can be used to treat cells in vitro, and the modified cells can be optionally administered to patients (i.e., ex vivo). Conventional virus-based systems can include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Retrovirus, lentivirus, and adeno-associated virus gene transfer methods can be integrated into the host genome, typically resulting in long-term expression of the inserted transgene. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors have very high transduction efficiencies in many cell types and do not require cell division.

[0374] In some embodiments, administration includes administration by other non-viral delivery of nucleic acid. Exemplary non-viral delivery methods include but are not limited to RNP complexes, lipofection, nuclear transfection, microinjection, gene guns, virions, liposomes, LNPs, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake. Lipofection is described in, for example, U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355), and lipofection reagents are commercially available (e.g., Transfectam TM and Lipofectin TM ). Cationic and neutral lipids suitable for effective receptor recognition polynucleotide lipofection include those of Feign...

Claims

1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20.

2. The nucleic acid molecule of claim 1, wherein the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided sequence-specific manner when the RGN polypeptide is bound to a guide RNA (gRNA) that is capable of hybridizing to a non-target strand of a target sequence, wherein the target sequence comprises a target strand and a non-target strand.

3. The nucleic acid molecule of claim 1 or 2, wherein the polynucleotide encoding the RGN polypeptide is operably linked to a promoter that is heterologous to the polynucleotide.

4. The nucleic acid molecule of any one of claims 1-3, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-20.

5. The nucleic acid molecule of any one of claims 1-4, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding.

6. The nucleic acid molecule of claim 5, wherein the RGN polypeptide is capable of producing a double-strand break.

7. The nucleic acid molecule of claim 5, wherein the RGN polypeptide is capable of producing single-strand breaks.

8. The nucleic acid molecule of any one of claims 1-4, wherein the RGN polypeptide is nuclease-inactive or is a nickase.

9. The nucleic acid molecule of any one of claims 1-8, wherein the RGN polypeptide is operably fused to a base editing polypeptide.

10. The nucleic acid molecule of claim 9, wherein the base editing polypeptide is a deaminase.

11. The nucleic acid molecule of any one of claims 1-10, wherein the RGN polypeptide comprises one or more nuclear localization signals.

12. The nucleic acid molecule of any one of claims 1-11, wherein the RGN polypeptide is codon-optimized for expression in a eukaryotic cell.

13. The nucleic acid molecule of any one of claims 1-12, wherein the target sequence is located adjacent to a protospacer adjacent motif (PAM). A vector comprising the nucleic acid molecule according to any one of claims 1 to 13.

15. The vector of claim 14, further comprising at least one nucleotide sequence encoding the gRNA, wherein the gRNA is capable of hybridizing to the non-target strand of the target sequence.

16. The vector of claim 15, wherein the guide RNA is selected from the group consisting of: a) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 42; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; b) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 43; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2; c) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 44 or to nucleotides 19-111 of SEQ ID NO: 1040; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3; d) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 24; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4; e) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; f) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:26; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 47 or to nucleotides 24-138 of SEQ ID NO: 1043; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6; g) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO: 1044 or nucleotides 27-95 of SEQ ID NO: 1045; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7; h) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:28; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 49; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8; i) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 29; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 50; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9; j) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO:51; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10; k) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 31; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO:52; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11; 1) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 32; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 53; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12; m) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 33; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO:54; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13; n) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 34; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO:55; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14; o) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 35; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO:56; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15; p) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 36; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 57; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16; q) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO:58; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; r) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 38 or 39; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 59 or 60; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18; s) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:40; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 61; wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19; and t) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:41; and ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 62; Wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

20.

17. The vector of claim 13, wherein the guide RNA is selected from the group consisting of: a) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 21; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 42; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 1; b) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 22; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 43; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:2; c) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 23; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 44 or to nucleotides 19-111 of SEQ ID NO: 1040; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:3; d) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:24; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 45; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:4; e) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:5; f) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:26; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 47 or to nucleotides 24-138 of SEQ ID NO: 1043; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:6; g) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO: 1044 or nucleotides 27-95 of SEQ ID NO: 1045; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:7; h) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:28; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 49; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:8; i) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:29; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 50; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:9; j) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 30; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 51; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 10; k) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 31; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 52; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 11; 1) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 32; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 53; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 12; m) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 33; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 54; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 13; n) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 34; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 55; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 14; o) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 35; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 56; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 15; p) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 36; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 57; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 16; q) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 37; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO:58; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 17; r) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 38 or 39; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 59 or 60; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 18; s) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:40; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 61; wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 19; and t) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:41; and ii) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 62; Wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:

20.

18. The vector of any one of claims 14-17, wherein the gRNA is a single guide RNA.

19. The vector of any one of claims 15-17, wherein the gRNA is a dual-guide RNA.

20. A cell comprising the nucleic acid molecule of any one of claims 1-14 or the vector of any one of claims 14-19.

21. A plant comprising the cell of claim 20.

22. A seed comprising the cell of claim 20.

23. A method of producing a RGN polypeptide, comprising culturing the cell of claim 20 under conditions whereby the RGN polypeptide is expressed.

24. A method of making an RGN polypeptide comprising introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; and culturing the cell under conditions where the RGN polypeptide is expressed.

25. The method of claim 24, wherein the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided, sequence-specific manner when the RGN polypeptide is bound to a guide RNA (gRNA) that is capable of hybridizing to a non-target strand of a target sequence, wherein the target sequence comprises a target strand and a non-target strand.

26. The method of claim 24 or 25, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-20.

27. The method of any one of claims 24-26, further comprising purifying the RGN polypeptide.

28. The method of any one of claims 24-27, wherein the cells further express one or more guide RNAs capable of binding to the RGN polypeptide to form a RGN ribonucleoprotein complex.

29. The method of claim 28, further comprising purifying the RGN ribonucleoprotein complex.

30. An RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20.

31. The RGN polypeptide of claim 31, wherein the RGN polypeptide is capable of binding to a target sequence in a target nucleic acid molecule in an RNA-guided, sequence-specific manner when the RGN polypeptide is bound to a guide RNA (gRNA) that is capable of hybridizing to a non-target strand of a target sequence, wherein the target sequence comprises a target strand and a non-target strand.

32. The RGN polypeptide of claim 30 or 31, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-20.

33. The RGN polypeptide of any one of claims 30-32, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule upon binding.

34. The RGN polypeptide of claim 33, wherein cleavage of the RGN polypeptide produces a double-stranded break. E 35. The RGN polypeptide of E 33, wherein single-strand breaks are produced by cleavage of the RGN polypeptide.

36. The RGN polypeptide of any one of claims 30-32, wherein the RGN polypeptide is nuclease-inactive or is a nickase. E 37. The RGN polypeptide of any one of E 30-36, wherein the RGN polypeptide is operably fused to a base editing polypeptide.

38. The RGN polypeptide of claim 37, wherein the base editing polypeptide is a deaminase.

39. The RGN polypeptide of any one of claims 30-38, wherein the target sequence is located adjacent to a protospacer adjacent motif (PAM). E 40. The RGN polypeptide of any one of E 28-36, wherein the RGN polypeptide comprises one or more nuclear localization signals.

41. A ribonucleoprotein (RNP) complex comprising the RGN polypeptide of any one of claims 30-40 and a guide RNA bound to the RGN polypeptide.

42. A nucleic acid molecule comprising a CRISPR RNA (crRNA) or a polynucleotide encoding a crRNA, wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, wherein the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NOs: 21-41, or nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045.

43. The nucleic acid molecule of claim 42, wherein the guide RNA comprises: a) the crRNA; and b) a trans-activating CRISPR RNA (tracrRNA) that hybridizes to the CRISPR repeat sequence of the crRNA; When the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide, the guide RNA is able to hybridize to a non-target strand of a target sequence in a target nucleic acid molecule through the spacer sequence of the crRNA in a sequence-specific manner.

44. The nucleic acid molecule of claim 42 or 43, wherein the polynucleotide encoding the crRNA is operably linked to a promoter heterologous to the polynucleotide.

45. The nucleic acid molecule of any one of claims 42-44, wherein the CRISPR repeat sequence comprises a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 21-41, or nucleotides 1-17 of SEQ ID NOs: 1041 or 1042, or nucleotides 1-22 of SEQ ID NOs: 1044 or 1045.

46. ​​A vector comprising a nucleic acid molecule comprising the polynucleotide encoding the crRNA described in any one of claims 42-45.

47. The vector of claim 46, wherein the vector further comprises a polynucleotide encoding the tracrRNA.

48. The vector of claim 47, wherein the tracrRNA is selected from the group consisting of: a) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 42, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 21; b) a tracrRNA having at least 90% sequence identity to SEQ ID NO:43, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:22; c) a tracrRNA having at least 90% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:23; d) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 45, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 24; e) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042; f) a tracrRNA having at least 90% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:26; g) a tracrRNA that has at least 90% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO: 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NOs: 1044 or 1045; h) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 49, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 28; i) a tracrRNA having at least 90% sequence identity to SEQ ID NO:50, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:29; j) a tracrRNA having at least 90% sequence identity to SEQ ID NO:51, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:30; k) a tracrRNA having at least 90% sequence identity to SEQ ID NO:52, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:31; 1) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 53, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 32; m) a tracrRNA having at least 90% sequence identity to SEQ ID NO:54, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:33; n) a tracrRNA having at least 90% sequence identity to SEQ ID NO:55, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:34; o) a tracrRNA having at least 90% sequence identity to SEQ ID NO:56, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:35; p) a tracrRNA having at least 90% sequence identity to SEQ ID NO:57, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:36; q) a tracrRNA having at least 90% sequence identity to SEQ ID NO:58, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:37; r) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 59 or 60, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 38 or 39; s) a tracrRNA having at least 90% sequence identity to SEQ ID NO:61, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:40; and t) a tracrRNA having at least 90% sequence identity to SEQ ID NO:62, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:

41.

49. The vector of claim 47, wherein the tracrRNA is selected from the group consisting of: a) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 42, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 21; b) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 43, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 22; c) a tracrRNA having at least 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:23; d) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 45, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 24; e) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042; f) a tracrRNA having at least 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:26; g) a tracrRNA that has at least 100% sequence identity to SEQ ID NO: 48, or to nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 27, or to nucleotides 1-22 of SEQ ID NOs: 1044 or 1045; h) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 49, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 28; i) a tracrRNA having at least 100% sequence identity to SEQ ID NO:50, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:29; j) a tracrRNA having at least 100% sequence identity to SEQ ID NO:51, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:30; k) a tracrRNA having at least 100% sequence identity to SEQ ID NO:52, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:31; 1) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 53, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 32; m) a tracrRNA having at least 100% sequence identity to SEQ ID NO:54, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:33; n) a tracrRNA having at least 100% sequence identity to SEQ ID NO:55, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:34; o) a tracrRNA having at least 100% sequence identity to SEQ ID NO:56, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:35; p) a tracrRNA having at least 100% sequence identity to SEQ ID NO:57, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:36; q) a tracrRNA having at least 100% sequence identity to SEQ ID NO:58, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:37; r) a tracrRNA having at least 100% sequence identity to SEQ ID NO: 59 or 60, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 38 or 39; s) a tracrRNA having at least 100% sequence identity to SEQ ID NO:61, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:40; and t) a tracrRNA having at least 100% sequence identity to SEQ ID NO:62, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:

41. E 50. The vector of any one of E 42-49, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide.

51. The vector of claim 50, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 1, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 21, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 42; b) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:2, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:22, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:43; c) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:3, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:23, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:4, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:24, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:45; e) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:5, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042; f) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:6, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:26, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:7, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NO:1044 or 1045, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO:1045; h) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:8, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:28, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:49; i) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:9, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:29, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:50; j) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 10, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 30, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 51; k) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 11, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 31, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 52; 1) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 32, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 53; m) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 13, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 33, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 54; n) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 14, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 34, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 55; o) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 15, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 35, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 56; p) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 16, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 36, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 57; q) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 17, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 37, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 58; r) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 18, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 38 or 39, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 59 or 60; s) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 19, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 40, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 61; and t) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:20, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:41, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:

62.

52. The vector of claim 50, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 1, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 21, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 42; b) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:2, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:22, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:43; c) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:3, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:23, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:4, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:24, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:45; e) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:5, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042; f) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:6, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:26, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:7, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NO:1044 or 1045, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:48 or to nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO:1045; h) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:8, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:28, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:49; i) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:9, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:29, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:50; j) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 10, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 30, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 51; k) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 11, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 31, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 52; 1) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 32, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 53; m) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 13, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 33, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 54; n) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 14, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 34, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 55; o) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 15, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 35, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 56; p) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 16, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 36, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 57; q) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 17, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 37, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 58; r) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 18, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 38 or 39, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 59 or 60; s) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 19, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO: 40, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 61; and t) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:20, wherein the CRISPR repeat sequence has at least 100% sequence identity to SEQ ID NO:41, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:

62.

53. A nucleic acid molecule comprising a trans-activating CRISPR RNA (tracrRNA) or a polynucleotide encoding a tracrRNA, wherein the polynucleotide encoding the tracrRNA comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NOs: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045.

54. The nucleic acid molecule of claim 53, wherein the guide RNA comprises: a) the tracrRNA; and b) a crRNA comprising a spacer sequence and a CRISPR repeat sequence, wherein the tracrRNA hybridizes to the CRISPR repeat sequence of the crRNA; When the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide, the guide RNA is able to hybridize to a non-target strand of a target sequence in a target nucleic acid molecule through the spacer sequence of the crRNA in a sequence-specific manner.

55. The nucleic acid molecule of claim 53 or 54, wherein the polynucleotide encoding the tracrRNA is operably linked to a promoter heterologous to the polynucleotide.

56. The nucleic acid molecule of any one of claims 53-55, wherein the tracrRNA comprises a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 42-62, nucleotides 19-111 of SEQ ID NO: 1040, nucleotides 22-85 of SEQ ID NOs: 1041 or 1042, nucleotides 24-138 of SEQ ID NO: 143, nucleotides 27-96 of SEQ ID NO: 1044, or nucleotides 27-95 of SEQ ID NO: 1045.

57. A vector comprising a nucleic acid molecule comprising a polynucleotide encoding the tracrRNA of any one of claims 53-56.

58. The vector of claim 57, wherein the vector further comprises a polynucleotide encoding the crRNA.

59. The vector of claim 58, wherein the crRNA comprises a CRISPR repeat sequence selected from the group consisting of: a) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:21, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:42; b) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:22, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:43; c) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:23, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:24, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:45; e) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; f) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:26, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045; h) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:28, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:49; i) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:29, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:50; j) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:30, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:51; k) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:31, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:52; l) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:32, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:53; m) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:33, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:54; n) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:34, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:55; o) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:35, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:56; p) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:36, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:57; q) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:37, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:58; r) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 38 or 39, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO: 59 or 60; s) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:40, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:61; and t) a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:41, wherein the tracrRNA has at least 90% sequence identity to SEQ ID NO:

62.

60. The vector of claim 58, wherein the crRNA comprises a CRISPR repeat sequence selected from the group consisting of: a) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:21, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:42; b) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:22, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:43; c) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:23, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:24, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:45; e) a CRISPR repeat sequence that has at least 100% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; f) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:26, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) a CRISPR repeat sequence that has at least 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045; h) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:28, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:49; i) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:29, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:50; j) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 30, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO: 51; k) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:31, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:52; l) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:32, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:53; m) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:33, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:54; n) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:34, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:55; o) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:35, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:56; p) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:36, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:57; q) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:37, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:58; r) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 38 or 39, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO: 59 or 60; s) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:40, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:61; and t) a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:41, wherein the tracrRNA has at least 100% sequence identity to SEQ ID NO:

62. E 61. The vector of any one of E 53-60, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide.

62. The vector of claim 61, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 1, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 42; b) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 2, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 43; c) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:3, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:23, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:4, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:24, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:45; e) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 5, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; f) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:6, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:26, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 7, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045; h) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:8, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:28, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:49; i) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:9, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:29, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:50; j) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 10, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 30, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 51; k) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 11, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 31, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 52; 1) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 12, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 32, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 53; m) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 13, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 33, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 54; n) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 14, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 34, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 55; o) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 15, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 35, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 56; p) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 16, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 36, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 57; q) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 17, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 37, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 58; r) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 18, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 38 or 39, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 59 or 60; s) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 19, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 40, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 61; and t) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:20, wherein the crRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:41, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:

62.

63. The vector of claim 61, wherein the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 1, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 21, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 42; b) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 2, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 22, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 43; c) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:3, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:23, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:4, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:24, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:45; e) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 5, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 25 or to nucleotides 1-17 of SEQ ID NO: 1041 or 1042, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 46 or to nucleotides 22-85 of SEQ ID NO: 1041 or 1042; f) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:6, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:26, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 7, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27 to 95 of SEQ ID NO: 1045; h) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:8, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:28, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:49; i) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:9, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:29, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:50; j) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 10, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 30, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 51; k) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 11, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 31, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 52; 1) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 12, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 32, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 53; m) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 13, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 33, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 54; n) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 14, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 34, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 55; o) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 15, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 35, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 56; p) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 16, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 36, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 57; q) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 17, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 37, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 58; r) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 18, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 38 or 39, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 59 or 60; s) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO: 19, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 40, and the tracrRNA has at least 100% sequence identity to SEQ ID NO: 61; and t) an RGN polypeptide having at least 100% sequence identity to SEQ ID NO:20, wherein the crRNA comprises a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:41, and the tracrRNA has at least 100% sequence identity to SEQ ID NO:

62.

64. A cell comprising the nucleic acid molecule of any one of claims 42-45 and 53-56, the vector of any one of claims 46-52 and 57-63, the single guide RNA of claim 142, or the dual guide RNA of claim 143.

65. A plant comprising the cell of claim 64.

66. A seed comprising the cell of claim 64.

67. A system for binding a target sequence in a target nucleic acid molecule, wherein the target sequence comprises a target strand and a non-target strand, the system comprising: a) one or more guide RNAs capable of hybridizing to the non-target strand of the target sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20, or a polynucleotide comprising a nucleotide sequence encoding said RGN polypeptide; The one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target sequence.

68. The system of claim 67, wherein at least one of the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the RGN polypeptide is operably linked to a promoter that is heterologous to the nucleotide sequence.

69. A system for binding a target sequence in a target nucleic acid molecule, wherein the target sequence comprises a target strand and a non-target strand, the system comprising: a) one or more guide RNAs capable of hybridizing to the non-target strand of the target sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; The one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target sequence.

70. The system of any one of claims 67-69, wherein at least one of the nucleotide sequences encoding the one or more guide RNAs is operably linked to a promoter that is heterologous to the nucleotide sequence.

71. The system of any one of claims 67-70, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-20.

72. The system of any one of claims 67-71, wherein the RGN polypeptide and the one or more guide RNAs are not found complexed with each other in nature.

73. The system of any one of claims 67-72, wherein the target sequence is a eukaryotic target sequence.

74. The system of any one of claims 67-73, wherein the gRNA is a single guide RNA (sgRNA).

75. The system of any one of claims 67-73, wherein the gRNA is a dual-guide RNA.

76. The system of any one of claims 67-75, wherein the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1; b) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:43, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2; c) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3; d) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:45, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4; e) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; f) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:26 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6; g) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; h) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:50, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:30 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:51, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:10; k) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:31 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:52, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:11; l) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:32 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:53, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12; m) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:33 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:54, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:13; n) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:34 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:55, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:14; o) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:35 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:56, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:15; p) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:36 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:57, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:16; q) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:37 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:58, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:17; r) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 59 or 60, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18; s) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:40 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:61, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:19; and t) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:62, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

20.

77. The system of any one of claims 67-75, wherein the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:1; b) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:43, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:2; c) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:3; d) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:45, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:4; e) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:5; f) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:26 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:6; g) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 7; h) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:50, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:30 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:51, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:10; k) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:31 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:52, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:11; l) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:32 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:53, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:12; m) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:33 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:54, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:13; n) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:34 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:55, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:14; o) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:35 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:56, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:15; p) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:36 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:57, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:16; q) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:37 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:58, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:17; r) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 59 or 60, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 18; s) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:40 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:61, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:19; and t) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:62, wherein the RGN polypeptide comprises an amino acid sequence having at least 100% sequence identity to SEQ ID NO:

20.

78. The system of any one of claims 67-77, wherein the target sequence is located adjacent to a protospacer adjacent motif (PAM).

79. The system of any one of claims 67-78, wherein the target sequence is intracellular.

80. The system of any one of claims 67-81, wherein the one or more guide RNAs are capable of hybridizing to the non-target strand of the target sequence, and the guide RNAs are capable of forming a complex with the RGN polypeptide to guide cleavage of the target nucleic acid molecule.

81. The system of claim 80, wherein the cleavage produces a double-strand break.

82. The system of claim 80, wherein the cleavage produces a single strand break.

83. The system of any one of claims 67-79, wherein the RGN polypeptide is nuclease-inactive or is a nickase.

84. The system of any one of claims 67-83, wherein the RGN polypeptide is operably linked to a base editing polypeptide.

85. The system of claim 84, wherein the base editing polypeptide is a deaminase. E 86. The system of any one of E 67-85, wherein the RGN polypeptide comprises one or more nuclear localization signals. E 87. The system of any one of E 67-86, wherein the RGN polypeptide is codon-optimized for expression in a eukaryotic cell.

88. The system of any one of claims 67-87, wherein the system further comprises one or more donor polynucleotides.

89. A cell comprising the system of any one of claims 67-88.

90. A plant comprising the cell of claim 89.

91. A seed comprising the cell of claim 89.

92. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-13, 42-45 and 53-56, the vector of any one of claims 14-19, 46-52 and 57-63, the cell of any one of claims 20, 64 and 89, the RGN polypeptide of any one of claims 30-40, the RNP complex of claim 41 or the system of any one of claims 67-88, and a pharmaceutically acceptable carrier.

93. A method for binding a target sequence in a target nucleic acid molecule, comprising delivering the system according to any one of claims 67-88 to the target sequence or a cell comprising the target sequence.

94. The method of claim 93, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target sequence.

95. The method of claim 93, wherein the guide RNA or the RGN polypeptide further comprises an expression regulator, thereby regulating the expression of a target gene comprising the target sequence.

96. A method for cleaving and / or modifying a target nucleic acid molecule comprising a target sequence, the method comprising delivering a system according to any one of claims 67-88 to the target sequence or a cell comprising the target sequence, wherein cleavage or modification of the target nucleic acid molecule occurs.

97. The method of claim 96, wherein the modified target nucleic acid molecule comprises an insertion of heterologous DNA into the target nucleic acid molecule.

98. The method of claim 96, wherein the modified target nucleic acid molecule comprises a deletion of at least one nucleotide in the target nucleic acid molecule.

99. The method of claim 96, wherein the modified target nucleic acid molecule comprises a mutation of at least one nucleotide in the target nucleic acid molecule.

100. A method for binding a target sequence in a target nucleic acid molecule, wherein the target sequence comprises a target strand and a non-target strand, the method include: a) assembling an RNA-guided nuclease (RGN) ribonucleotide complex under conditions suitable for forming the RGN ribonucleotide complex by combining: i) one or more guide RNAs capable of hybridizing to the non-target strand of the target sequence; and ii) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; as well as b) contacting the target nucleic acid molecule or a cell comprising the target nucleic acid molecule with the assembled RGN ribonucleotide complex; wherein the one or more guide RNAs hybridize to the non-target strand of the target sequence, thereby directing the RGN polypeptide to bind to the target sequence.

101. The method of claim 100, wherein the method is performed in vitro, in vivo, or ex vivo.

102. The method of claim 100 or 101, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target sequence.

103. The method of claim 100 or 101, wherein the guide RNA or the RGN polypeptide further comprises an expression regulator, thereby allowing for said regulating expression of a target gene comprising the target sequence.

104. The method of claim 100 or 101, wherein the RGN polypeptide further comprises a base editing polypeptide, thereby allowing modification of the target nucleic acid molecule.

105. The method of claim 104, wherein the base editing polypeptide comprises a deaminase.

106. The method of claim 100 or 101, wherein the RGN polypeptide is capable of cleaving the target nucleic acid molecule, thereby allowing for cleavage and / or modification of the target nucleic acid molecule.

107. A method for cleaving and / or modifying a target nucleic acid molecule comprising a target sequence, wherein the target sequence comprises a target strand and a non-target strand, the method comprising contacting the target nucleic acid molecule with: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20; and b) one or more guide RNAs capable of targeting the RGN of (a) to the target sequence; The one or more guide RNAs hybridize to the non-target strand of the target sequence, thereby guiding the RGN polypeptide to bind to the target nucleic acid molecule, and cleavage and / or modification of the target nucleic acid molecule occurs.

108. The method of claim 107, wherein double-strand breaks are produced by cleavage of the RGN polypeptide.

109. The method of claim 107, wherein single-strand breaks are produced by cleavage of the RGN polypeptide.

110. The method of claim 107, wherein the RGN polypeptide is nuclease-inactive or a nickase and is operably fused to a base editing polypeptide.

111. The method of claim 110, wherein the base editing polypeptide is a deaminase.

112. The method of claim 107, wherein the modified target nucleic acid molecule comprises an insertion of heterologous DNA into the target nucleic acid molecule.

113. The method of claim 107, wherein the modified target nucleic acid molecule comprises a deletion of at least one nucleotide in the target nucleic acid molecule.

114. The method of claim 107, wherein the modified target nucleic acid molecule comprises a mutation of at least one nucleotide in the target nucleic acid molecule.

115. The method of any one of claims 106-113, wherein the target sequence is located adjacent to a protospacer adjacent motif (PAM).

116. The method of any one of claims 107-115, wherein the target sequence is a eukaryotic target sequence.

117. The method of any one of claims 107-116, wherein the gRNA is a single guide RNA (sgRNA).

118. The method of any one of claims 107-116, wherein the gRNA is a dual-guide RNA. E 119. The method of any one of E 107-118, wherein the RGN comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-20.

120. The method of any one of claims 107-118, in: a) the RGN has at least 90% sequence identity to SEQ ID NO: 1, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 21 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 42; b) the RGN has at least 90% sequence identity to SEQ ID NO:2, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:43; c) the RGN has at least 90% sequence identity to SEQ ID NO:3, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) the RGN has at least 90% sequence identity to SEQ ID NO:4, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:45; e) the RGN has at least 90% sequence identity to SEQ ID NO:5, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042, and a tracrRNA having at least 90% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042; f) the RGN has at least 90% sequence identity to SEQ ID NO:6, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:26 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) the RGN has at least 90% sequence identity to SEQ ID NO:7, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NO:1044 or 1045, and a tracrRNA having at least 90% sequence identity to SEQ ID NO:48 or nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO:1045; h) the RGN has at least 90% sequence identity to SEQ ID NO:8, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:49; i) the RGN has at least 90% sequence identity to SEQ ID NO:9, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:50; j) the RGN has at least 90% sequence identity to SEQ ID NO: 10, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 51; k) the RGN has at least 90% sequence identity to SEQ ID NO: 11, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 31 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 52; l) the RGN has at least 90% sequence identity to SEQ ID NO: 12, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 32 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 53; m) the RGN has at least 90% sequence identity to SEQ ID NO: 13, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 33 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 54; n) the RGN has at least 90% sequence identity to SEQ ID NO: 14, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 34 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 55; o) the RGN has at least 90% sequence identity to SEQ ID NO: 15, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 35 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 56; p) the RGN has at least 90% sequence identity to SEQ ID NO: 16, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 36 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 57; q) the RGN has at least 90% sequence identity to SEQ ID NO: 17, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58; r) the RGN has at least 90% sequence identity to SEQ ID NO: 18, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 59 or 60; s) the RGN has at least 90% sequence identity to SEQ ID NO: 19, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 40 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 61; and t) the RGN has at least 90% sequence identity to SEQ ID NO:20, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:

62.

121. The method of any one of claims 107-118, in: a) the RGN has at least 100% sequence identity to SEQ ID NO: 1, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 21 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 42; b) the RGN has at least 100% sequence identity to SEQ ID NO:2, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:43; c) the RGN has at least 100% sequence identity to SEQ ID NO:3, the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040; d) the RGN has at least 100% sequence identity to SEQ ID NO:4, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:45; e) the RGN has at least 100% sequence identity to SEQ ID NO:5, the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042, and a tracrRNA having at least 100% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042; f) the RGN has at least 100% sequence identity to SEQ ID NO:6, the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:26 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043; g) the RGN has at least 100% sequence identity to SEQ ID NO:7, the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:27 or to nucleotides 1-22 of SEQ ID NO:1044 or 1045, and a tracrRNA having at least 100% sequence identity to SEQ ID NO:48 or to nucleotides 27-96 of SEQ ID NO 1044 or to nucleotides 27-95 of SEQ ID NO:1045; h) the RGN has at least 100% sequence identity to SEQ ID NO:8, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:49; i) the RGN has at least 100% sequence identity to SEQ ID NO:9, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:50; j) the RGN has at least 100% sequence identity to SEQ ID NO: 10, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 30 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 51; k) the RGN has at least 100% sequence identity to SEQ ID NO: 11, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 31 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 52; 1) the RGN has at least 100% sequence identity to SEQ ID NO: 12, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 32 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 53; m) the RGN has at least 100% sequence identity to SEQ ID NO: 13, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 33 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 54; n) the RGN has at least 100% sequence identity to SEQ ID NO: 14, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 34 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 55; o) the RGN has at least 100% sequence identity to SEQ ID NO: 15, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 35 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 56; p) the RGN has at least 100% sequence identity to SEQ ID NO: 16, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 36 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 57; q) the RGN has at least 100% sequence identity to SEQ ID NO: 17, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 37 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 58; r) the RGN has at least 100% sequence identity to SEQ ID NO: 18, the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 59 or 60; s) the RGN has at least 100% sequence identity to SEQ ID NO: 19, the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity to SEQ ID NO: 40 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 61; and t) the RGN has at least 100% sequence identity with SEQ ID NO:20, and the guide RNA comprises a crRNA repeat sequence having at least 100% sequence identity with SEQ ID NO:41 and a tracrRNA having at least 100% sequence identity with SEQ ID NO:

62.

122. The method of any one of claims 107-121, wherein the target sequence is intracellular.

123. The method of claim 122, further comprising culturing the cells under conditions where the RGN polypeptide is expressed, and cleaving and modifying the target nucleic acid molecule to produce a DNA molecule comprising the modified target nucleic acid molecule; and selecting cells comprising the modified target nucleic acid molecule.

124. A cell comprising a modified target nucleic acid molecule according to the method of claim 123.

125. A plant comprising the cell of claim 124.

126. A seed comprising the cell of claim 124.

127. A pharmaceutical composition comprising the cell of claim 124 and a pharmaceutically acceptable carrier.

128. A method for producing a genetically modified cell having a correction in a causal mutation of a genetically inherited disease, the method comprising introducing into the cell: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20, or a polynucleotide encoding the RGN polypeptide, wherein the polynucleotide encoding the RGN polypeptide is operably linked to a promoter such that the RGN polypeptide can be expressed in the cell; and b) a guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable the gRNA to be expressed in the cell, The RGN and gRNA thereby target the genomic location of the causal mutation and modify the genomic sequence to remove the causal mutation.

129. The method of claim 128, wherein the RGN is nuclease-inactive or is a nickase and is fused to a polypeptide having base editing activity.

130. The method of claim 129, wherein the base editing polypeptide is a deaminase.

131. The method of any one of claims 128-129, wherein the genetic inherited disease is caused by a single nucleotide polymorphism.

132. The method of claim 131, wherein the gRNA further comprises a spacer sequence targeting a proximal region of the causal single nucleotide polymorphism.

133. A method for producing a genetically modified cell having a deletion in a pathogenic expanded trinucleotide repeat sequence, the method comprising introducing into the cell: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-20, or a polynucleotide encoding the RGN polypeptide, wherein the polynucleotide encoding the RGN polypeptide is operably linked to a promoter such that the RGN polypeptide can be expressed in the cell; and b) a first guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell, and further wherein the gRNA comprises a spacer sequence targeting the 5' flank of the amplified trinucleotide repeat sequence; and c) a second guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell, and further wherein the second gRNA comprises a spacer sequence targeting the 3' flank of the amplified trinucleotide repeat sequence; Thereby the RGN and the two gRNAs are targeted to the expanded trinucleotide repeat sequence, and at least a portion of the expanded trinucleotide repeat sequence is removed.

134. The method of claim 133, wherein the first gRNA further comprises a spacer sequence targeting a region within the amplified trinucleotide repeat sequence or a proximal region of the amplified trinucleotide repeat sequence.

135. The method of claim 134, wherein the second gRNA further comprises a spacer sequence targeting a region within the amplified trinucleotide repeat sequence or a proximal region of the amplified trinucleotide repeat sequence. E 136. The method of any one of E 133-135, wherein the RGN polypeptide has at least 100% sequence identity to any one of SEQ ID NOs: 1-20.

137. The method of any one of claims 133-136, wherein the gRNA, the first gRNA, the second gRNA, or the first gRNA and the second gRNA are selected from a gRNA selected from the group consisting of: a) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1; b) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:43, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2; c) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3; d) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:45, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4; e) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; f) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:26 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6; g) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; h) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:50, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:30 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:51, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:10; k) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:31 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:52, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:11; l) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:32 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:53, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12; m) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:33 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:54, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:13; n) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:34 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:55, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:14; o) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:35 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:56, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:15; p) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:36 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:57, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:16; q) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:37 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:58, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:17; r) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 58 or 60, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18; s) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:40 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:61, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:19; and t) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:62, wherein the RGN polypeptide has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

20.

138. The method of any one of claims 133-136, wherein the gRNA, the first gRNA, the second gRNA, or the first gRNA and the second gRNA are selected from a gRNA selected from the group consisting of: a) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:42, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:1; b) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:43, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:2; c) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:44 or to nucleotides 19-111 of SEQ ID NO:1040, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:3; d) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:45, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:4; e) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:25 or to nucleotides 1-17 of SEQ ID NO:1041 or 1042 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:46 or to nucleotides 22-85 of SEQ ID NO:1041 or 1042, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:5; f) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:26 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:47 or to nucleotides 24-138 of SEQ ID NO:1043, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:6; g) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 27 or to nucleotides 1-22 of SEQ ID NO: 1044 or 1045 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 48 or to nucleotides 27-96 of SEQ ID NO 1044 or nucleotides 27-95 of SEQ ID NO: 1045, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 7; h) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:28 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:49, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:29 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:50, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:30 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:51, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:10; k) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:31 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:52, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:11; l) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:32 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:53, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:12; m) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:33 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:54, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:13; n) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:34 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:55, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:14; o) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:35 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:56, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:15; p) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:36 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:57, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:16; q) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:37 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:58, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:17; r) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO: 38 or 39 and a tracrRNA having at least 100% sequence identity to SEQ ID NO: 58 or 60, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 18; s) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:40 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:61, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:19; and t) a gRNA comprising a CRISPR repeat sequence having at least 100% sequence identity to SEQ ID NO:41 and a tracrRNA having at least 100% sequence identity to SEQ ID NO:62, wherein the RGN polypeptide has an amino acid sequence having at least 100% sequence identity to SEQ ID NO:

20.

139. A method of treating a disease, disorder, or condition, the method comprising administering the pharmaceutical composition of claim 92 or 127 to a subject in need thereof.

140. The method of claim 139, wherein the disease, disorder or condition is associated with a causal mutation and the pharmaceutical composition corrects the causal mutation.

141. The method of claim 139 or 140, wherein the subject is at risk for developing the disease, disorder, or condition.

142. A single guide RNA comprising the nucleic acid molecule containing the crRNA of any one of claims 42-45 and the nucleic acid molecule containing the tracrRNA of any one of claims 53-57.

143. A dual guide RNA comprising the nucleic acid molecule containing the crRNA of any one of claims 42-45 and the nucleic acid molecule containing the tracrRNA of any one of claims 53-57.

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